Immunomodulating compositions and methods of use thereof

ABSTRACT

This invention is directed to β-1-6-glucans, compositions and devices comprising the same, and methods of use thereof in modulating immune responses. The β-1-6-glucans of certain embodiments of the invention are enriched for O-acetylated groups and/or conjugated to a solid support or linked to a targeting moiety.

GOVERNMENT INTEREST STATEMENT

This invention was made in whole or in part with government supportunder Grant Number GM035010-22 awarded by the National Institutes ofHealth. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

The cell walls of fungi evoke a powerful immuno-stimulatory response,and have been proposed for use as potential anti-infective andanti-tumor drugs. Fungal cells can also activate dendritic cells andprime class II-restricted antigen-specific T cell responses. Themajority of the cell wall (50-60%) of pathogenic (Candida albicans) andnon-pathogenic fungi (Saccharomvces cerevisiae) is composed of an innerlayer of 3-glucan (β-1,3- and β-1,6-glucan) covalently linked to avariety of cell surface mannoproteins [Klis, F. M. et al. et al. MedMycol 39 Suppl 1, 1-8, 2001; Klis, F. M. et al. et al., FEMS MicrobiolRev 26, 239-56, 2002].

Recognition of β-glucans by macrophages is carried out mainly throughDectin-1 with the cooperation of TLRs, including TLR2 [Brown, G. D. etal. et al. Nature 413, 36-7, 2001]. Dectin-1 activity is inhibited byβ-1,3-glucans and β-1,6-glucans, with the β-1,3-glucan laminarins havingthe highest effect. However, oligosaccharide microarray results showthat Dectin-1 binds specifically to β-1,3-glucans. Neutrophils areprofessional killers, whose role in phagocytosis and killing of bacteriaand fungi is well characterized. Neutropenic individuals are much moresusceptible to bacterial and fungal infections, with return to normalcounts playing an important role in resolution of infection.Neutrophils, unlike macrophages, require serum for optimal phagocytosisand killing. The main opsonic receptors are the complement receptor CR3and the immunoglobulin-binding receptor FcγR. CR3 has a lectin domain[Brown, G. D. et al. Immunity 19, 311-5, 2003] that mediates increasedneutrophil motility towards a mixture of β-1,3-glucan and β-1,6-glucan(PGG-glucan) [Wakshull, E. et al. Immunopharmacology 41, 89-107, 1999].Although neutrophils express Dectin-1 [Taylor, P. R. et al. J Immunol169, 3876-82, 2002], its role in fungal recognition is not yet clear.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, a composition comprisingβ-1-6-glucan enriched for O-acetylated groups. In one embodiment, theglucan contains at least 25% by weight O-acetylated glucan. In oneembodiment, the glucan is isolated or derived from lichen, which, in oneembodiment, is from the genus Umbilicariaceae. In one embodiment, theglucan is isolated from a fungus. In one embodiment, the glucan isisolated from yeast, or in another embodiment the glucan is chemicallysynthesized or acetylated. In another embodiment, the glucan isconjugated to a particle.

In another embodiment, the composition comprises an adjuvant, anantigen, an immuno-modulatory compound, or a combination thereof.

In another embodiment, this invention provides a method of modulating animmune response in a subject, said method comprising administering tosaid subject a composition comprising β-1-6-glucan enriched forO-acetylated groups.

According to this aspect of the invention, and in one embodiment,modulating the immune response comprises stimulating said immuneresponse, which, in one embodiment, is an antigen-specific response. Inone embodiment, the composition further comprises an immuno-stimulatorycompound, or in another embodiment, a chemotherapeutic compound. Inanother embodiment, the immune response is directed against aninfectious agent, a cancer or other type of tumor, a pre-neoplasticlesion or a combination thereof. In another embodiment, the immuneresponse is not directed against a cancer or other type of tumor.

In another embodiment, modulating the immune response comprisesdown-modulating or abrogating the immune response. According to thisaspect, and in one embodiment, the composition further comprises animmunosuppressant. In one embodiment, the immune response is directedagainst an autoantigen or in another embodiment, an allergen, or inanother embodiment, the immune response is directed against transplantedtissue or in another embodiment, transplanted cells. In one embodiment,the composition is administered to a subject suffering from anautoimmune disorder. In one embodiment the autoimmune disorder isassociated with excessive neutrophil activity, neutrophil infiltration,neutrophil degranulation, etc. In one embodiment the disorder is adisorder that affects the skin; the composition may be applied directlyto the skin.

In another embodiment this invention provides a composition comprisingβ-1-6-glucan, wherein the glucan is conjugated to a particle. Accordingto this aspect, and in one embodiment, the glucan is enriched forO-acetylated groups, and in one embodiment, contains at least 25% byweight O-acetylated glucan. In another embodiment, the glucan isisolated or derived from a lichen or a yeast. In one embodiment, theglucan is isolated or derived from Umbilicariaceae. In anotherembodiment, the glucan is chemically synthesized or acetylated. In oneembodiment, the glucan is conjugated to a microsphere, which, in oneembodiment, has a diameter of about 0.1-15 microns.

In another embodiment, the composition comprises an adjuvant, anantigen, an immuno-modulatory compound, or a combination thereof.

In another embodiment, this invention provides a method of modulating animmune response in a subject, the method comprising administering to thesubject a composition comprising β-1-6-glucan, wherein the glucan isconjugated to a solid support such as a particle.

In another embodiment, the invention provides a particle comprisingβ-1-6-glucan. In certain embodiments, the particle consists of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%β-1-6-glucan by dry weight. In certain embodiments, the particleconsists essentially of β-1-6-glucan. Optionally, in certainembodiments, the β-1-6-glucan is enriched for O-acetylated groups. Theinvention further provides a method of modulating the immune response ofa mammalian subject comprising administering any of the afore-mentionedparticles, or a composition containing any of the afore-mentionedparticles, to the subject.

In one embodiment, modulating the immune response comprises stimulatingthe immune response, which in one embodiment is an antigen-specificresponse. According to this aspect of the invention and in oneembodiment, the composition further comprises an immuno-stimulatorycompound or in another embodiment, a chemotherapeutic compound. In oneembodiment, the immune response is directed against an infectious agent,a cancer or other type of tumor, a preneoplastic lesion or a combinationthereof

In another embodiment, modulating the immune response comprisesdownmodulating or abrogating the immune response. According to thisaspect of the invention and in one embodiment, the composition furthercomprises an immunosuppressant. In one embodiment, the immune responseis directed against an autoantigen or, in another embodiment, anallergen, or in another embodiment, transplanted tissue or, in anotherembodiment, transplanted cells. In one embodiment, the composition isadministered to a subject suffering from an autoimmune disorder. In oneembodiment the autoimmune disorder is associated with excessiveneutrophil activity, neutrophil infiltration, neutrophil degranulation,etc. In one embodiment the disorder is a disorder that affects the skin,the composition may be applied directly to the skin.

In another embodiment, this invention provides a method of treating,delaying progression of, prolonging remission of, or reducing theincidence or severity of cancer in a subject, said method comprisingadministering to said subject a composition comprising purifiedβ-1-6-glucan. In another embodiment, this invention provides a method oftreating, delaying progression of, prolonging remission of, or reducingthe incidence or severity of a tumor in a subject, said methodcomprising administering to said subject a composition comprisingpurified β-1-6-glucan.

In one embodiment, the β-1-6-glucan is enriched for O-acetylated groups,which in one embodiment contains at least 25-% by weight O-acetylatedglucan. In another embodiment, the composition further comprises anadjuvant, an antigen, a peptide, an immuno-stimulatory compound, achemotherapeutic or a combination thereof.

In one embodiment, the antigen is a tumor-associated antigen, or inanother embodiment, the peptide is derived from a tumor-associatedantigen.

In one embodiment, the subject has a hyperplastic or preneoplasticlesion. In another embodiment, the subject has cancer. In anotherembodiment, the subject has not been diagnosed with cancer. In anotherembodiment, the subject has not been diagnosed with a tumor.

In another embodiment, this invention provides a method of treating,delaying progression of, or reducing the incidence or severity of aninfection in a subject, said method comprising administering to saidsubject a composition comprising purified β-1-6-glucan.

In one embodiment, the β-1-6-glucan is enriched for O-acetylated groups,which in one embodiment contains at least 25% by weight O-acetylatedglucan. In another embodiment, the composition further comprises anadjuvant, an antigen, a peptide, an immuno-stimulatory compound, achemotherapeutic or a combination thereof.

In one embodiment, the antigen or peptide is derived from the source ofthe infection. In one embodiment, the immuno-stimulatory compound is acytokine. In another embodiment, the chemotherapeutic compound is anantibiotic or antiviral compound. In one embodiment, the compositioncomprises a steroid. In another embodiment the composition comprises3-1,3 glucans having β-1,6-glucan branches (also referred to as beta1,3/1,6, glucan or beta-1,6-branched beta-1,3-glucan) wherein at leastsome of the β-1,6-glucan branches are enriched for O-acetylated groups.In another embodiment the invention provides a composition comprising(i) β-1-6-glucan enriched for O-acetylated groups; and (ii)beta-1,6-branched beta-1,3-glucan.

In one embodiment, the invention provides a food supplement comprisingβ-1-6-glucan enriched for O-acetylated groups. In one embodiment, theinvention provides a food product comprising β-1-6-glucan enriched forO-acetylated groups. In another embodiment, the invention provides acosmetic composition comprising β-1-6-glucan enriched for O-acetylatedgroups.

In another embodiment, this invention provides a method of inducingexpression of heat shock proteins in neutrophils, the method comprisingcontacting neutrophils with a composition comprising β-1-6-glucan,optionally enriched for O-acetylated groups. In another embodiment, thisinvention provides a method of inducing phagocytosis and production ofreactive oxygen species in neutrophils, the method comprising contactingneutrophils with a composition comprising β-1-6-glucan, optionallyenriched for O-acetylated groups. In another embodiment, this inventionprovides a method of inducing expression of heat shock proteins inneutrophils, the method comprising contacting neutrophils with acomposition comprising β-1-6-glucan, wherein at least 25% of the glucoseunits in at least 5% of the glucan molecules are enriched forO-acetylated groups.

In any of the afore-mentioned embodiments the contacting may occureither outside the body of a subject or within the body. In oneembodiment, cells, which in some embodiments are neutrophils, areremoved from a subject, contacted with the composition, and thenadministered to the subject at a later time. In one embodiment the cellsare contacted with the composition for a time sufficient to induceexpression of heat shock proteins. In certain embodiments the cells arealso contacted with serum or with one or more serum components. In oneembodiment the subject receives immunosuppressive therapy prior toadministration of the cells. For example, a subject may be in need ofimmunosuppressive therapy for organ transplantation or other purposes,e.g. chemotherapy or radiation therapy for cancer, leukemia, lymphoma,or any type of tumor, wherein the therapy would tend to render theindividual immunocompromised. In one embodiment of the invention, priorto administering the immunosuppressive therapy, immune system cells areremoved from the subject. The cells (which, in some embodiments, areneutrophils or in other embodiments, other immune system cells, such asother professional antigen-presenting cells, such as macrophages,dendritic cells, monocytes, NK cells, B cells or others) are contactedoutside the body with a composition of this invention and are thenreturned to the subject a suitable period of time after the subject hasreceived the immunosuppressive therapy. The suitable period of timecould be, for example, after the therapy has been administered or itscytotoxic effects have diminished, when the subject is at risk of orexhibits symptoms or signs of infection, etc.

In one embodiment, the invention provides a method of inducingexpression of heat shock proteins in a subject comprising administeringβ-1-6-glucan, optionally enriched for O-acetylated glucan, to a subjectin an amount sufficient to induce expression of heat shock proteins incells, e.g. neutrophils, of the subject. In one embodiment, theinvention provides a method of inducing production of reactive oxygenspecies in a subject comprising administering β-1-6-glucan, optionallyenriched for O-acetylated glucan, to a subject in an amount sufficientto induce production of reactive oxygen species by cells, e.g.neutrophils, of the subject. In one embodiment, the invention provides amethod of enhancing phagocytosis in as subject comprising administeringβ-1-6-glucan, optionally enriched for O-acetylated glucan, to a subjectin an amount sufficient to enhance phagocytosis by cells, e.g.neutrophils, of the subject.

The invention further provides a coated material comprising (a) asubstrate; and (b) a compound or composition comprising β-1-6-glucan. Incertain embodiments the β-1-6-glucan is enriched for O-acetylatedglucan. In certain embodiments the coated material comprises a coatinglayer, e.g. a gel or a film, having the β-1-6-glucan physicallyassociated therewith. In certain embodiments the coating layer comprisesa polymer, e.g. an organic polymer, in addition to the β-1,6-glucan,wherein the polymer is physically associated with the β-1,6-glucan. Insome embodiment the polymer is covalently bound to the β-1,6-glucanwhile in other embodiments the polymer is mixed with or impregnated withthe β-1,6-glucan. In certain embodiments the coating layer containsbetween 1% and 90% β-1,6-glucan by dry weight. In certain embodimentsthe coating layer contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% β-1,6-glucan by dry weight. In certain embodiments thecoating layer comprises less than 10%, 20%, 30%, 40%, 50% β-1,6-glucanby dry weight.

A variety of different polymers are of use in the invention. Somesuitable polymers are disclosed herein. In certain embodiments thepolymer is biodegradable. In such embodiments the compound orcomposition may be released as the polymer degrades. In some embodimentsthe polymer is a poly (pyranose), poly(hydroxyl acid), poly(lactone),poly (amino acid), poly(anhydride), poly (urethane), poly (orthoester),poly (phosphazine), poly(phosphoester), poly (lactic) acid, poly(glycolic) acid, poly (lactic-co-glycolic) acid, poly(ether ester),poly(amino acid), synthetic poly(amino acid), polycarbonate,poly(hydroxyalkanoate), poly(ε-caprolactone), or poly(saccharide), or amixture or blend of any of the foregoing. In certain embodiments thepolymer is a copolymer, which in certain embodiments is a blockcopolymer, wherein the subunits are subunits found in any of theforegoing polymers. In certain embodiments a polymer of use in theinvention has an average molecular weight (e.g. a number average orweight average molecular weight) of at least 10, 25, 50, 100, 200, or300 kD, or a value falling within any intervening range. In certainembodiments a polymer of use in the invention is composed of, on averagebetween 100 and 10,000 monomeric subunits, or any number of subunitsfalling within an intervening range. In certain embodiments the polymeris selected from polyethylene, polypropylene, polyvinyl chloride,polyethylene terephthalate, polystyrene and polycarbonate.

In one embodiment, the substrate is a part of, or in the form of amicroparticle, nanoparticle, bandage, suture, catheter, stent, valve,pacemaker, implantable defibrillator, conduit, cannula, appliance,scaffold, central line (which may be a peripherally inserted centralcatheter (PICC or PIC line)), pessary, tube, drain, shunt, trochar,plug, or other implant or medical or surgical device. In one embodiment,the catheter is a pulmonary artery, pericardial, pleural, urinary orintra-abdominal catheter. In one embodiment, the drain is acerebrospinal fluid drain. In one embodiment, the tube is atracheostomy, endotracheal or chest tube. In another embodiment, thesubstrate is a part of, or in the form of an implant, a rod (e.g. aspinal rod such as a posterior spinal rod), a plate, a screw, washer,wire, pin, internal fixation devices (e.g. fracture fixation devices),or other implantable orthopedic hardware known to those of skill in theart.

Also provided by the present invention are methods of using the implantor other device. The implants and devices may be used in any manner inwhich conventional counterparts (e.g. counterparts not comprising and/orcoated with a compound or composition disclosed herein) are used, suchmethods being known in the art. In some embodiments, a method of thisinvention is to be understood as comprising the treatment of any diseaseor condition with the implant or device of the invention. Also providedby the present invention are methods of delivering a compound orcomposition disclosed herein comprising a β-1,6-glucan to a subject,wherein the method comprises implanting or introducing a coatedmaterial, implant, or other device comprising a compound or compositionof the invention into the body of the subject. Also provided by thepresent invention are methods of delivering a therapeutic agent notcomprising a β-1,6-glucan to a subject using an implant or other device(e.g. a catheter, implantable pump, indwelling intravenous line, etc.)that comprises a β-1,6-glucan.

In another embodiment, this invention provides a composition comprisinga β-1-6-glucan physically associated with a targeting moiety, forexample one that specifically interacts with or attracts a phagocyticcell. According to this aspect of the invention and in one embodiment,the targeting moiety specifically interacts with an infected cell, aneoplastic cell, a pre-neoplastic cell, a pathogen or a componentthereof, or is one that recruits phagocytic cells, for example, to sitesof neoplasia, preneoplasia, infection, etc. According to this aspect ofthe invention and in one embodiment, the targeting moiety specificallyinteracts with an infected cell, a neoplastic cell, a pre-neoplasticcell, a pathogen, or a component thereof. In one embodiment, the glucanis enriched for O-acetylated groups, and in one embodiment, the glucancontains at least 25% by weight O-acetylated glucan. In anotherembodiment, the glucan is isolated or derived from a lichen or a yeast,which in one embodiment is Umbilicariaceae. In one embodiment, theglucan is chemically synthesized or acetylated. In another embodiment,the composition further comprises an adjuvant, an antigen, animmuno-modulatory compound, or a combination thereof. In anotherembodiment, the phagocytic cell is a professional antigen-presentingcell. In another embodiment, the phagocytic cell is a neutrophil.

In one embodiment, the targeting moiety is an antibody or antibodyfragment.

In another embodiment, the invention provides a method modulating animmune response in a subject, the method comprising administering to thesubject a composition comprising a β-1-6-glucan physically associatedwith a targeting moiety, for example, wherein the targeting moietyspecifically interacts with or attracts a phagocytic cell, or comprisesany embodiment as herein described.

In one embodiment, modulating said immune response comprises stimulatingsaid immune response, which in one embodiment is an antigen-specificresponse. In one embodiment, the composition further comprises animmuno-stimulatory compound, or in another embodiment, the compositionfurther comprises a chemotherapeutic compound. In one embodiment, theimmune response is directed against an infectious agent, a cancer, apre-neoplastic lesion or a combination thereof, and in anotherembodiment, the immune response is complement-dependent.

In one embodiment, this invention provides a method of treating,delaying progression of, or reducing the incidence or severity of aninfection in a subject, said method comprising administering to saidsubject a composition comprising a β-1-6-glucan physically associatedwith a targeting moiety, for example, wherein the targeting moietyspecifically interacts with or attracts a phagocytic cell, or comprisesany embodiment as herein described. In one embodiment, the compositionfurther comprises an adjuvant, an antigen, a peptide, animmuno-stimulatory compound, a chemotherapeutic or a combinationthereof. In one embodiment, the antigen or peptide is derived from thesource of the infection. In another embodiment, the immuno-stimulatorycompound is a cytokine. In another embodiment, the chemotherapeuticcompound is an antibiotic or antiviral compound.

In one embodiment, this invention provides a method of stimulating orenhancing heat shock protein expression in a cell, the method comprisingcontacting the cell with a composition comprising a β-1-6-glucanphysically associated with a targeting moiety, for example, wherein thetargeting moiety specifically interacts with or attracts a phagocyticcell, or comprises any embodiment as herein described.

In another embodiment, this invention provides a method of modulating animmune response in a subject, said method comprising administering tosaid subject a composition comprising β-1-6-glucan, wherein said glucanis conjugated to a particle, as herein described.

In another embodiment, this invention provides a method of treating,delaying progression of, prolonging remission of, or reducing theincidence or severity of cancer in a subject, said method comprisingadministering to said subject a composition comprising a glucan asherein described, for example, a purified β-1-6-glucan, a preparationenriched for O-acetylated β-1-6-glucan, a β-1-6-glucan conjugated to aparticle or a β-1-6-glucan linked to a targeting moiety or a combinationthereof.

In another embodiment, this invention provides a micelle comprisingβ-1-1,6-glucan, wherein said β-1,6-glucan is optionally enriched forO-acetylated glucan. In another embodiment, this invention provides acomposition comprising β-1,6-glucan and a biodegradable polymer, whereinsaid biodegradable polymer degrades to form biologically activesalicylate or alpha-hydroxy acid moieties and said β-1,6-glucan isoptionally enriched for O-acetylated glucan. In another embodiment, thisinvention provides for the use of any glucan, any composition, anymicelle or combination thereof for any method as herein described.

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. In case of a conflict between thespecification and an incorporated reference, the specification shallcontrol. Where number ranges are given in this document, endpoints areincluded within the range. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges, optionally including or excluding either or bothendpoints, in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. Where a percentage is recited in reference to avalue that intrinsically has units that are whole numbers, any resultingfraction may be rounded to the nearest whole number.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. β-1,6-glucan stimulates expression of heat shock proteins (HSPs)in neutrophils. Induction of HSPs was determined by quantitativereal-time PCR. The data represent the average of two (A) or at leastthree experiments with standard deviation. Candida (Ca) or beads wereopsonized with pooled human serum and cultured for 2 hours withneutrophils. (A) Candida albicans elicits HSPs in neutrophils. The foldinduction represents the ratio of neutrophils+Candida to neutrophilsalone. (B) Heat-killed Candida elicits higher levels of HSPs. Resultsfor heat-killed Candida were normalized to UV-killed Candida. (C-E)β-1,6-glucan stimulates expression of HSPs. Polybead polystyrene 6.0micron microspheres (beads) were coated with an equivalent amount of oneof the following glucans: laminarin (lam, algal β-1,3-glucan),β-1,6-glucan purified from Candida albicans (Ca β-1,6-glucan), pustulan(pus, lichen β-1,6-glucan), barley glucan (bar, 30% β-1,3-glucan, and70% β-1,4-glucan), or dextran (dex, α-1,6-glucan). Beads were opsonizedwith pooled human serum, or with heat inactivated (HI) pooled humanserum (D). The fold induction represents the ratio ofneutrophils+glucans-coated beads over neutrophils+untreated beads. (C)Fungal β-1,6-glucan stimulates expression of HSPs. Beads were coatedwith β-1,3-glucan purified from Candida albicans (Ca β-1,3-glucan), orβ-1,6-glucan purified from Candida albicans (Ca β-1,6-glucan). (D)Standard β-1,6-glucan stimulates expression of HSPs. Beads were coatedwith laminarin (lam, algal β-1,3-glucan), or pustulan (pus, lichenβ-1,6-glucan). (E) Soluble β-1,6-glucan and other glucan-coated beads donot stimulate expression of HSPs. Beads were coated with β-glucanisolated from barley (bar, 30% β-1,3-glucan, and 70% β-1,4-glucan), orwith dextran (dex, α-1,6-glucan). Neutrophils were cultured with 5 mg/mlof soluble laminarin (sol lam), 5 mg/ml of soluble pustulan (sol pus),or beads. The fold induction represents the ratio of neutrophils+solubleglucans over neutrophils alone, or neutrophils+glucans-coated beads overneutrophils+untreated beads.

FIG. 2. Elicitation of HSPs by pustulan is due to β-1,6-glucan.Neutrophils were cultured with opsonized beads for 2 hours at 37° C. inA and F. (A) Endo-β-1,6-glucanase reduces the induction of HSPs bypustulan. Beads were coated with an equivalent amount of pustulan (pus)or endo-β-1,6-glucanase digested pustulan. Induction of HSPs with enzymetreated pustulan is relative to that with untreated. The data representthe average of several experiments with standard deviation. (B) Pustulanchromatographed on Biogel P6 column. (C) Pustulan digested first withendo-β-1,6-glucanase and run on a P6 column generated a large and asmall peak. The small peak represents a tiny fraction of the originalpustulan that was resistant to enzymatic digestion (Vo). (D) The largepeak in C was shown by thin layer chromatography to be the expecteddegradation products, gentiobiose and gentiotriose. Lane 1 containsstandard oligosaccharides (G to G5) as controls. Lane 2 is pustulanspiked with glucose (G). Lane 3 is endo-β-1,6-glucanase digested pus.Ori=origin. (E) Chromatography of deacetylated pustulan. The insert isan overlay of the Vo from C and E. (F) Deacetylation of pustulanfollowed by digestion with endo-β-1,6-glucanase eliminates induction ofHSPs. Induction of HSPs in deacetylated pustulan or deacetylatedpustulan digested with endo-β-1,6-glucanase is relative to that withuntreated pustulan.

FIG. 3. β-1,6-glucan stimulates phagocytosis and production of reactiveoxygen species (ROS) in neutrophils. Polybead polystyrene 6 μmmicrospheres (beads) were coated with an equivalent amount of theindicated β-glucans and then opsonized. (A) β-1,6-glucan stimulatesphagocytosis. Phagocytosis was assessed by time-lapse microscopy forbeads that were coated with laminarin (a, and b), or pustulan (c and d).The images at a and c were taken at time 0. Images b and d were takenafter culturing with neutrophils for 40 minutes. (B) β-1,6-glucanstimulates phagocytosis. Phagocytosis was assessed byFluorescence-Activated Cell Sorting (FACS) by the change in side scatterfor neutrophils with (a) untreated beads, (b) beads coated withβ-1,3-glucan from Candida (c) beads coated with laminarin (lam,β-1,3-glucan), (d) beads coated with glucan from barley (bar), (e) beadscoated with pustulan (pus, β-1,6-glucan) (f) soluble pustulan. (C)β-1,6-glucan stimulates ROS production. ROS production was assayed byFACS using DHR123. β-1,3-glucan shows only a modest stimulation.

FIG. 4. C3 proteolytic fragments are deposited on β-1,6-glucan. Beadswere untreated (Beads), or coated with equivalent amount of laminarin(lam, β-1,3-glucan), or pustulan (pus, β-1,6-glucan). Followingopsonization, the beads were suspended in 2% SDS 1M ammonium hydroxidebuffer and incubated at 37° C. for 1 hour. The supernatant solution wasloaded on 4-20% acrylamide SDS gel. The migration of the molecularweight protein standards is indicated. (A) The gel was incubated withsilver stain, and the bands were extracted for analysis by massspectrometry. (B) Western analysis was performed using monoclonalantibodies directed against (a) the alpha or (b) the beta chains of C3.

FIG. 5. Preincubation of serum with soluble pustulan (β-1,6-glucan)abolishes stimulation of neutrophils, whereas soluble laminarin(β-1,3-glucan) does not. (A) Phagocytosis of pustulan-coated beads wasassessed by FACS, by the change in side scatter. Serum was untreated(a), or incubated for 5 minutes at 37° C. with 1 mg (quantified byphenol-sulfuric acid method) of soluble laminarin (lam) (b) or pustulan(pus) (c). (B) Reactive oxygen species production in response topustulan-coated beads was assayed by FACS using DHRI23. Serum wasuntreated (red), incubated with soluble laminarin (green), or withpustulan (blue). (C) C3 deposition on pustulan-coated beads waseliminated by preincubation of the serum by soluble pustulan but notlaminarin. C3 deposition was assayed by Western analysis usingmonoclonal antibodies directed against the (a) alpha or (b) the betachains of C3. Serum was preincubated with soluble pustulan (1),laminarin (2), or was untreated (3). The molecular weight proteinstandard is indicated. (D) Preincubation of serum with soluble pustulanreduces Candida killing. Serum was untreated, or preincubated withsoluble pus or lam prior to opsonization of Candida. Candida viabilitywas assayed using XTT following incubation of 30 minutes withneutrophils.

FIG. 6. CR3 mediates β-1,6-glucan stimulation of neutrophils. Polybeadpolystyrene 6.0 micron microspheres (beads) were coated with theβ-1,6-glucan pustulan (pus). Beads were opsonized with pooled humanserum and cultured with neutrophils for 15 minutes. Neutrophils werepreincubated with CR3 blocking antibodies or IgG isotype control for 30minutes on ice before culturing with pustulan-coated beads. (A) CR3blocking antibodies reduce β-1,6-glucan-stimulated phagocytosis.Phagocytosis of pustulan-coated beads was assessed by FACS by the changein side scatter for neutrophils preincubated with: (a) isotype controlIgG, or (b) anti-CR3 blocking antibodies. (B) CR3 blocking antibodiesreduce β-1,6-glucan-stimulated ROS production. ROS production inresponse to pustulan-coated beads was assayed by FACS using DHRI23.Neutrophils were preincubated with isotype control IgG (green) or antiCR3 blocking Ab (red).

FIG. 7. β-1,6-glucan elicits chemokines in monocytes. Polybeadpolystyrene 6.0 micron microspheres (beads) were coated with equivalentamount of the β-1,3-glucan laminarin (lam), or the β-1,6-glucan pustulan(pus). Beads were opsonized with pooled human serum. Monocytes werecultured for 2 hours with 5 mg/ml of soluble lam or pus, or with thebeads described above. Induction of chemokines was determined byquantitative real-time PCR. Results were averaged and standarddeviations were calculated.

FIG. 8. Schematic depiction of pustulan-antibody chimera activity incellular targeting and complement deposition, leading to neutrophilengulfment. Antibody (Ab) is physically linked to polysaccharide (PS).Complement (C3) deposition recruits neutrophils to target cells as afunction of the antibody specificity

FIG. 9. Elicitation of high reactive oxygen species by human neutrophilspost-exposure to pustulan-anti-Candida albicans monoclonal antibodychimeras. Anti-C. albicans antibody (Ab)-pustulan (pus) (Ab-pus)complexes 30-100 kDa or higher than 100 kDa (>100) in size were loadedon polyacrylamide gels and silver-stained (A). ROS production wasassayed by Fluorescence-Activated Cell Sorting using DHR123 (B).

FIG. 10. β-1,6-glucan is required for efficient phagocytosis of Candidaalbicans, production of ROS, and expression of HSPs. Candida albicanscells were heat-killed, digested with an endo-β-1,6-glucanase, andopsonized. (A) β-1,6-glucan is required for efficient phagocytosis.Phagocytosis was assessed by Fluorescence-Activated Cell Sorting (FACS)by the change in side scatter. (B) β-1,6-glucan is required forefficient ROS production. ROS production was assayed by FACS usingDHR123. (C) β-1,6-glucan is required for induction of HSPs. HSPsinduction was determined by quantitative real-time PCR. Results forβ-1,6-glucanase digested Candida were normalized to undigested Candida.The data represent the average of two experiments with standarddeviation.

FIG. 11. (A) Mouse serum activates complement. Beads were untreated (1),or coated with equivalent amount of pustulan (β-1,6-glucan) (2) orlaminarin (β-1,3-glucan) (3). Following opsonization with mouse(C57Black/6) serum, the beads were suspended in 2% SDS 1M ammoniumhydroxide buffer and incubated at 37° C. for 1 hour. The supernatantsolution was loaded on 4-20% acrylamide SDS gel. The migration of themolecular weight protein standards is indicated. Western analysis wasperformed using anti-mouse C3 antibodies. (B) β-1,6-glucan-coated beadsprotect mice from systemic fungal infection. Candida albicans cells(10⁶) were injected into the tail vein of C57Black/6 mice. 10⁵β-1,6-glucan-coated beads or untreated beads were injected the next dayinto the tail vein of the same mice. Survival was monitored daily.

FIG. 12. PLGA beads encapsulating β-1,6-glucan elicit production ofreactive oxygen species and protect mice from systemic fungal infection.PLGA beads were made with 250 mg polysaccharide per mg of PLGA. (A) SEMimages of PLGA beads (a,d) or PLGA beads encapsulating β-1,3-glucan(b,e) or β-1,6-glucan (c,f) at day 0 (a-c) or after 3 days (d-f). (B)β-1,6-glucan is detected on the surface of degrading PLGA beads.β-1,6-glucan was detected on degrading PLGA beads following 3 days ofincubation in PBS, using polyclonal anti-β-1,6-glucan antibodies. (C)PLGA beads encapsulating β-1,6-glucan (7.17 microgram glucose/mg ofPLGA) (green) elicit higher levels of reactive oxygen species than PLGAbeads encapsulating β-1,3-glucan (37.1 microgram glucose/mg of PLGA)(red). (D) PLGA beads encapsulating β-1,6-glucan protect mice fromsystemic fungal infection. Candida albicans cells (10⁶) were injectedinto the tail vein of C57Black/6 mice. 10⁵ PLGA beads encapsulatingβ-1,6-glucan beads, PLGA beads encapsulating β-1,3-glucan or PLGA beadswere injected the next day into the tail vein of the same mice. Survivalwas monitored daily.

FIG. 13. High levels of IgG are detected on β-1,6-glucan. Beads wereuntreated (red), or coated with equivalent amount of laminarin(β-1,3-glucan) (green) or pustulan (β-1,6-glucan) (blue). Followingopsonization, IgM and IgG deposition was detected using anti-human IgMand IgG antibodies.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity. Further, where consideredappropriate, reference numerals may be repeated among the figures toindicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

Glucans are polysaccharides found in all studied species of lichenizedfungi. Partially O-acetylated pustulans are typical of Umbilicariaceae,and have been described for several species of Umbilicaria, such as U.pustulata and U. hirsute, U. angulata, U. caroliniana, and U.polyphylla.

β-1,6-glucans, were found to induce specific gene expression in contrastto β-1,3-glucans, which activity may be useful in modulating immuneresponses. Without intending any limitation, O-acetylated β-1,6-glucanswere found to be useful in this context. Without intending anylimitation, β-1,6-glucans were also found to induce phagocytosis andreactive oxygen species production by neutrophils. Reactive oxygenspecies are an important component of the killing mechanism inneutrophils, and therefore, this activity of β-1,6-glucan may be usefulin modulating immune responses. In one embodiment, the inventionprovides a method of inducing production of reactive oxygen species in asubject comprising administering β-1-6-glucan, optionally enriched forO-acetylated glucan, to a subject in an amount sufficient to inducephagocytosis and production of reactive oxygen species by cells, e.g.neutrophils, of the subject. Reactive oxygen species (ROS) includemolecules such as oxygen ions, free radicals and peroxides bothinorganic and organic. In certain embodiments they are small moleculesand are highly reactive due to the presence of unpaired valence shellelectrons. In one embodiment, the ROS is superoxide.

This invention provides a composition comprising purified β-1-6-glucan,wherein the composition is, in various embodiments of the invention, apharmaceutical composition, a food or food product, a food supplement,or a cosmetic composition. The composition is, in some embodiments,distinct from compositions such as pustulan or preparations of fungalcell walls. In certain embodiments of the invention at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of the glucancontained in the composition by weight is β-1-6-glucan. In certainembodiments between 20% and 50% of the glucan contained in thecomposition is β-1-6-glucan. In certain embodiments between 50% and 100%of the glucan contained in the composition is β-1-6-glucan. In oneembodiment of any of the compositions or methods of the invention, theglucan contains from about 15% to about 30% by weight β-1-6-glucan. Inanother embodiment of any of the compositions or methods of theinvention, the glucan contains from about 10% to about 35% by weightβ-1-6-glucan, or in another embodiment, from about 20% to about 50% byweight β-1-6-glucan, or in another embodiment, from about 25% to about60% by weight β-1-6-glucan, or in another embodiment, from about 35% toabout 80% by weight β-1-6-glucan, or in another embodiment, from about18% to about 35% by weight β-1-6-glucan, or in another embodiment, fromabout 15% to about 75% by weight β-1-6-glucan. In certain embodiments ofthe invention “weight” refers to “dry weight”. In other embodiments“weight” refers to total weight. In certain embodiments of the inventionthe β-1-6-glucan is processed. Such processing may comprise, forexample, deacetylation, treatment with enzymes that digest glucans otherthan β-1-6-glucan, limited digestion with enzymes that digestβ-1-6-glucan, selection of particular molecular weight ranges, etc. Incertain embodiments, processing comprises separation from other glucans,e.g. β-glucans, β-1-3 glucans, etc. In certain embodiments theprocessing comprises removing β-1-6-glucan side chains from β-1-3glucans and optionally separating the β-1-6-glucans side chains. Incertain embodiments the composition comprises processed β-1-6-glucan,wherein the processed β-1-6-glucan exhibits enhanced ability todesirably modulate the immune response relative to unprocessed glucan orrelative to unprocessed β-1-6-glucan.

This invention provides, in one embodiment, a composition comprisingβ-1-6-glucan enriched for O-acetylated groups. In one embodiment of anyof the compositions or methods of the invention, the glucan contains atleast 25% by weight O-acetylated glucan. In one embodiment of any of thecompositions or methods of the invention, the glucan contains from about15% to about 30% by weight O-acetylated glucan. In another embodiment ofany of the compositions or methods of the invention, the glucan containsfrom about 10% to about 35% by weight O-acetylated glucan, or in anotherembodiment, from about 20% to about 50% by weight O-acetylated glucan,or in another embodiment, from about 25% to about 60% by weightO-acetylated glucan, or in another embodiment, from about 35% to about80% by weight O-acetylated glucan, or in another embodiment, from about18% to about 35% by weight O-acetylated glucan, or in anotherembodiment, from about 15% to about 75% by weight O-acetylated glucan.In other embodiments, the glucan contains between about 75% and 100% byweight O-acetylated glucan, e.g. between 75% and 90%, or between 90% and100% by weight O-acetylated glucan. In one embodiment of any of thecompositions or methods of the invention the glucan containsapproximately that percentage of O-acetylated glucose units (by weightor number, in various embodiments of the invention) that would resultfrom digestion of a naturally occurring β-1-6-glucan (e.g. pustulan orany other β-1-6-glucan mentioned herein) with a β-1-6 endoglucanase fora time sufficient to digest at least 90% by weight of the β-1-6-glucanto oligosaccharides comprising 5 or fewer glucose units followed by (i)removal of those oligosaccharides comprising 5 or fewer glucose residuesfrom the composition or (ii) isolation of a portion of the resultingcomposition having a molecular weight greater than 5 kD, or in someembodiment greater than 10, 20, 30, 50, or 100 kD.

In some embodiments, the term “enriched for O-acetylated residues”refers to the enhanced % of O-acetylated sites in individual glucoseunits within the glucan molecule, enhanced % of O-acetylated glucoseunits within the glucan molecule, or a combination thereof, as comparedto a native glucan molecule. In one embodiment, reference to glucanpreparations enriched by a particular weight percent for O-acetylatedglucan, refers to preparations comprising an enhanced % of O-acetylatedsites in individual glucose units within the glucan molecule, anenhanced % of O-acetylated glucose units within the glucan molecule, ora combination thereof, as compared to a glucan molecule.

Glucans derived from different sources may comprise varying amounts ofO-acetylation in terms of O-acetylated sites in individual glucoseunits, O-acetylated glucose units within the glucan molecule, or acombination thereof. According to this aspect of the invention, the term“enriched for O-acetylated glucan” refers, in some embodiments, toenhanced O-acetylation as described herein, between the reference sourcefrom which the glucan is derived, and may not represent an overallenrichment as compared to any glucan source.

In one embodiment, the term “enriched for O-acetylated glucan” refers,to an enrichment of at least 25% by weight of the glucan chains, whichare O-acetylated on at least one glucose unit, or at least 25% of theglucose units present in the glucan in the composition are O-acetylated,or a combination thereof. In some embodiments, at least 25% of theglucose units in at least 1%, or in another embodiment, at least 5% ofthe beta glucan chains are O-acetylated. In other embodiments between25% and 35%, between 25% and 50%, between 25% and 75%, between 15% and45%, between 20% and 60%, between 35% and 80%, or others of the glucoseunits in at least 5% of the beta glucan chains are O-acetylated, etc. Inother embodiments, embodiments between 25% and 35%, between 25% and50%/o, between 25% and 75%, between 15% and 45%, between 20% and 60%,between 35% and 80%, or others of the glucose units, in at least 10% ofthe beta glucan chains, or in another embodiment, in at least 15% of thebeta glucan chains, or in another embodiment, in at least 20% of thebeta glucan chains, are O-acetylated.

In one embodiment, the glucan is isolated or derived from a lichen,which in one embodiment is from the genus Umbilicariaceae. In oneembodiment, the glucan is isolated from a fungus. In one embodiment, thefungus is an edible mushroom, inter alia, Grifola frondosa (maitake),Cordyceps sinensis, Agaricus brasiliensis, Inonotus obliquus (Chaga)).In one embodiment, the glucan is isolated from yeast, or in anotherembodiment the glucan is chemically synthesized or acetylated. In oneembodiment, short synthetic β-1,6-glucan polymers are linked throughlinkers (e.g. diamine) to form long polymers. In another embodiment, theglucan is conjugated to a solid support.

Glucans are glucose-containing polysaccharides found, inter alia, infungal cell walls. α-glucans include one or more α-linkages betweenglucose subunits and β-glucans include one or more β-linkages betweenglucose subunits

β-1,6-glucans occur frequently in fungi but are rarer outside fungi. Theglucan used in accordance with the invention comprises β-1,6-glucan. Insome embodiments, the β-glucans are derived from Umbilicariaceae, suchas U. pustulata and U. hirsute, U. angulata, U. caroliniana, and U.polyphylla.

In some embodiments, the β-glucans are derived from Candida, such as C.albicans. Other organisms from which β-glucans may be used includeCoccidioides immitis, Trichophyton verrucosum, Blastomyces dermatidis,Cryptococcus neoformans, Histoplasma capsulatum, Saccharomycescerevisiae, Paracoccidioides brasiliensis, and Pythiumn insidiosum. Insome embodiments, the β-glucans are chemically or enzymaticallysynthesized, as is known in the art, or in other embodiments, theβ-glucans are derived from any species producing the same, andchemically or enzymatically altered, for example, to increaseO-acetylation of the molecule.

In some embodiments, the β-glucans are fungal glucans. A ‘fungal’ glucanwill generally be obtained from a fungus but, where a particular glucanstructure is found in both fungi and non-fungi (e.g. in bacteria, lowerplants or algae) then the non-fungal organism may be used as analternative source.

Full-length native β-glucans are insoluble and have a molecular weightin the megadalton range. In some embodiments, this invention providessoluble β-1,6-glucan. In some embodiments, this invention providessoluble O-acetylated β-1,6-glucan. Solubilization may be achieved byfragmenting long insoluble glucans, in some embodiments. This may beachieved by, for example, hydrolysis or, in some embodiments, bydigestion with a glucanase (e.g. with a β-1,3 glucanase or limiteddigestion with a β-1,3 glucanase). In other embodiments, glucans can beprepared synthetically, for example, and in some embodiments, by joiningmonosaccharide building blocks. O-acetylation of such glucans canreadily be accomplished by methods known in the art. Such methods mayinclude chemical and/or enzymatic acetylation, such as are known in theart.

There are various sources of fungal β-glucans. For instance, pureβ-glucans are commercially available e.g. pustulan (Calbiochem) is aβ-1,6-glucan purified from Umbilicaria papullosa. β-glucans can bepurified from fungal cell walls in various ways, for example, asdescribed in Tokunaka et al. [(1999) Carbohydr Res 316:161-172], and theproduct may be enriched for β-1,6-glucan moieties, or O-acetylatedβ-1,6-glucan moieties, by methods as are known in the art.

One of ordinary skill in the art will be able to identify or selectappropriate methods to enrich for β-1,6-glucan moieties and/or forO-acetylated β-1,6-glucan. In one embodiment, O-acetylation ofbeta-glucan is performed chemically. For example, polysaccharides (50mg) are dried in a speed vac centrifuge and resuspended in 1.5 ml ofacetic anhydride (Mallindcrockdt). After resuspension, a few crystals of4-dimethylaminopyridine (Avocado Research Chemist, Ltd) are added ascatalyst. The reaction is allowed to proceed at room temperature for 5,20, or 120 minutes and then stopped with 2 volumes of water. Afterwardsthe samples are dialyzed overnight against water. It will be appreciatedthat this process could be varied or scaled up, as evident to one ofskill in the art. In other embodiments, methods for separatingO-acetylated β-1,6-glucan include one or more of the following steps,which could be performed in various orders: (a) separation based onhigher hydrophobicity, such as binding to any hydrophobic matrix/resin;(b) separation based on digestion with a suitable endo- or exo-glucanaseor combination thereof, wherein the O-acetylated β-1,6-glucan isresistant to digestion; (c) affinity separation using antibodies orother moieties that bind to β-1,6-glucan or to O-acetyl groups thereon;(d) separation based on molecular weight. In one embodiment,β-1,6-glucan is digested with an enzyme that digests unacetylated and/orlightly acetylated β-1,6-glucan. The resulting material is separatedbased on size or molecular weight and a portion comprising heavilyacetylated glucan is isolated. In some embodiments, β-1,6-glucanpreparations are obtained, digested and O-acetylated oligosaccharidesare separated or in another embodiment, isolated, and used in thepreparation of new compositions. Such compositions represent embodimentsof the β-1,6-glucan preparations enriched for O-acetylated residues ofthis invention.

It is to be understood that the products of any process for preparingenriched O-acetylated β-1,6-glucan preparations are to be considered aspart of this invention.

In some embodiments, the glucans for use in the compositions,preparations, micelles and/or according to the methods of this inventionmay comprise structural modifications, not present in native glucanpreparations. Such modifications may comprise, O-acetylation, asdescribed herein. In other embodiments, such modifications may comprisemethylation, alkylation, alkoylation, sulfation, phosphorylation, lipidconjugation or other modifications, as are known to one skilled in theart. In some embodiments the modification comprises modification (e.g.esterification) with an acid such as formic, succinic, citric acid, orother acid known in the art.

In some embodiments, lipid conjugation to any or all free hydroxylgroups may be accomplished by any number of means known in the art, forexample, as described in Drouillat B, et al., Pharm Sci. 1998 January;87(1):25-30, B. N. A. Mbadugha, et al., Org. Lett., 5 (22), 4041-4044,2003.

In some embodiments, methylation may be accomplished and verified by anynumber of means known in the art, for example, as described in Mischnicket al. 1994 Carbohydr. Res., 264, 293-304; Bowie et al. 1984, Carbohydr.Res., 125, 301-307; Sherman and Gray 1992, Carbohydr. Res., 231,221-235; Stankowski and Zeller 1992, Carbohydr. Res., 234, 337-341;Harris, P. J., el al. (1984) Carbohydr. Res. 127, 59-73; Carpita, N. C.& Shea, E. M. (1989) Linkage structure of carbohydrates by gaschromatography-mass spectrometry (GC-MS) of partially methylated alditolacetates. In Analysis of Carbohydrates by GLC and MS (Biermann, C. J. &McGinnis, G. D., eds), pp. 157-216. CRC Press, Boca Raton, Fla.

In some embodiments, methylation can be confirmed by GLC offurther-derived TMS ethers, acetates or other esters, coupled MS, ordigestion to monosaccharides, de-O-methylation and analysis byderivatization and GLC/MS, for example as described in Pazur 1986,Carbohydrate Analysis—A Practical Approach, IRL Press, Oxford, pp.55-96; Montreuil et al. 1986, Glycoproteins. In M. F. Chaplin and J. F.Kennedy, (eds.), Carbohydrate Analysis—a Practical Approach, IRL Press,Oxford, pp. 143-204; Sellers el al. 1990, Carbohydr. Res., 207, C1-C5;O'Neill el al. 1990, Pectic polysaccharides of primary cell walls. In P.M. Dey (ed.), Methods in Plant Biochemistry, Volume 2, Carbohydrates,Academic Press, London, pp. 415-441; Stephen et al. 1990, Methods inPlant Biochemistry, Volume 2, Carbohydrates, Academic Press, London, pp.483-522; or Churms 1991, CRC Handbook of Chromatography. Carbohydrates,Volume II, CRC Press, Boca Raton, Fla., USA).

In some embodiments, phosphorylation, optionally including theintroduction of other modifications, and verification of the obtainedproduct may be accomplished by means well known to those skilled in theart, see for example, Brown, D. H., Biochem. Biophys. Acta, 7, 487(1951); Roseman, S., and Daffner, I., Anal. Chem., 28, 1743 (1956);Kornberg, A., and Horecker, B. L., in Methods in enzymology, Vol. 1,Academic Press, New York, 1955, p. 323; U.S. Pat. No. 4,818,752.

In some embodiments, glucan sulfation and verification of the obtainedproduct may be accomplished by any of the means well known in the art,for example, as described in Alban, S., and Franz, G. (2001),Biomacromolecules 2, 354-361; Alban, el al. (1992) Arzneimittelforschung42, 1005-1008; or Alban, S., et al. (2001). Carbohydr. Polym. 47,267-276.

Also provided by the invention is a micelle comprising β-1,6-glucan. Incertain embodiments the micelle comprises a complex composed ofsurfactant molecules comprising β-1,6-glucan, which may be dispersed ina liquid colloid. In certain embodiments the surfactant molecules areamphilic, i.e., they contain both hydrophobic groups (their “tails”) andhydrophilic groups (their “heads”). In certain embodiments thehydrophilic component comprises β-1,6-glucan, optionally modifiedaccording to any one or more ways described herein. In certainembodiments a micelle in aqueous solution forms an aggregate with thehydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic tail regions in the micelle center. Themicelle may be globular and roughly spherical in shape, but in certainembodiments the micelle is an ellipsoid, cylinder, or bilayer. In someembodiments the micelle is a polymeric micelle such as those describedin U.S. Pub. No. 20020035217. In some embodiments the micelleencapsulates an active agent, e.g. a hydrophobic molecule. Exemplaryactive agents include anti-infective agents such as anti-bacterial,anti-viral, anti-fungal, anti-parasite agents; chemotherapeutic agentsfor treatment of cancer; immunostimulatory compound, antigen, adjuvant,etc.

The invention further provides β-1,6-glucan that is modified byconjugating a lipid thereto, wherein the modification in someembodiments allows for creation of a micelle comprising β-1,6-glucanhaving the lipid attached thereto. The lipid may be a straight chain orbranched, optionally substituted, hydrocarbon. In some embodiments thelipid comprises a fatty acid. In some embodiments the lipid, e.g. fattyacid, contains between 4 and 26 or between 4 and 40 carbon atoms.

Also provided by the present invention is a particle comprisingβ-1,6-glucan covalently or noncovalently linked to a particle comprisingor consisting essentially of yeast glucan. Also provided is β-1,6-glucancomprising a reactive moiety able to react with a functional group of ayeast glucan to form a covalent bond. The yeast glucan may compriseβ-1,6-glucan, β-1,3-glucan, other glucans, or a combination thereof.

Also provided by the invention is a composition comprising β-1,6-glucanand a biodegradable polymer. In some embodiments the biodegradablepolymer comprises biologically active subunits. The term “biodegradable”refers to a material, which is degraded, i.e., broken down into smallerfragments, in the biological environment of the cell or subject in whichit is found. In one embodiment, biodegradation involves the degradationof a polymer into its component subunits, via, for example, enzymatic ornon-enzymatic hydrolysis, digestion, etc. In one embodiment,biodegradation may involve cleavage of bonds (whether covalent orotherwise) in the polymer backbone. In another embodiment,biodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to a side-chain or one that connects a side chain tothe polymer backbone. In some embodiments the degradation products aremetabolizable by the subject. In some embodiments the degradationproducts are usable by the subject for synthesis of larger biomolecules.In some embodiments the degradation products are excreted or otherwiseeliminated by the subject. In some embodiments the polymer and/or itsdegradation products are biocompatible in that they are substantiallynontoxic and do not produce an unacceptable inflammatory or immuneresponse when administered or otherwise introduced into the body of asubject in amounts consistent with the present invention.

In some embodiments, a biodegradable polymer encapsulating the glucansof this invention comprise particles of this invention. In someembodiments, such polymers may comprise poly(lactic-co-glycolic) acid(PLGA), hydrophobic bioabsorbable polymers such as polyglycolide,polylactide (D, L, DL), polydioxanones, polyestercarbonates,polyhydroxyalkonates, polycaprolactone (polylactones), polyethyleneglycol, hydroxypropylmethyl cellulose phthalate, cellulose acetatephthalate, and the Ludragit R, L, and E series of polymers and copolymermixtures thereof, and copolymers made from two or more precursors of theabove, prepared by any means known in the art, for example, as describedin U.S. Pat. Nos. 7,060,299, 6,998,115, 6,048,551, incorporated byreference herein in their entirety.

The term “biologically active agent” includes therapeutic agents thatprovide a therapeutically desirable effect when administered to ananimal (e.g. a mammal, such as a human) in effective amounts, it beingunderstood that not all subjects will benefit from the agent. In someembodiments the polymer is a polyanhydride, which optionally comprisesbiologically active salicylates and alpha-hydroxy acids. Degradation ofthe polymer releases said biologically active salicylates and/oralpha-hydroxy acids. In some embodiments the β-1,6-glucan is covalentlyor noncovalently attached to the biodegradable polymer. Suitablepolymers and methods for manufacture thereof are described, e.g. in U.S.Publication No. 20030035787 and 20050053577. In certain embodiments thepolymer comprises between 10 and 1000, or between 50 and 500, or about100 monomers. In one embodiment the polymer is polyaspirin®. Methods offorming a compound in which a β-1,6-glucan is covalently linked to thepolymer will be evident to one of skill in the art. The β-1,6-glucancould be covalently attached to a monomer prior to polymerization orcould be conjugated to a functional group of the polymer followingpolymerization. In some embodiments the β-1,6-glucan is covalentlyattached via a linking group. Exemplary linking groups are described inU.S. Pub. No. 20050053577, and others will be evident to one of skill inthe art.

In some embodiments the composition comprises a particle comprisingβ-1,6-glucan and the biodegradable polymer. In some embodiments theparticle is coated with or impregnated with β-1,6-glucan. In someembodiments the β-1,6-glucan is covalently attached to the polymer. Insome embodiments the composition coats an implant or other medical orsurgical device as described elsewhere herein.

Further provided are methods of administering a β-1,6-glucan and abiologically active salicylate or alpha-hydroxy acid to a subject, themethod comprising administering a composition comprising β-1,6-glucanand a biodegradable polymer comprising said biologically activesalicylates and/or alpha-hydroxy acids to the subject or implanting orintroducing a device comprising said polymer and said biologicallyactive salicylates and/or alpha-hydroxy acids into a subject.

In some embodiments, this invention provides low molecular weightglucans, having a molecular weight of less than 100 kDa (e.g. less than80, 70, 60, 50, 40, 30, 25, 20, or 15 kDa). In some embodiments, thisinvention provides oligosaccharides e.g. containing 85 or fewer (e.g.85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68,67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50,49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32,31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4) glucose monosaccharide units.

In some embodiments of any of the compositions, particles, coatedmaterials, or devices of the invention comprising β-1,6-glucan, theβ-1,6-glucan comprises or consists essentially of a low molecular weightglucan. In some embodiments of any method of the invention in whichβ-1,6-glucan is utilized, the β-1,6-glucan comprises or consistsessentially of a low molecular weight glucan. Optionally at least someof the low molecular weight β-1,6-glucan in any embodiment of theinvention is enriched for O-acetylated groups.

A common technique in determining linkage type and structure in glucansis carbon-13 nuclear magnetic resonance spectroscopy (¹³C-NMR). Thenumber and relative intensities of ¹³C signals in a given spectrum canbe used to determine linkage configurations and positions in glucanpolymers. For example, the chemical shifts (signals) of carbon atomsengaged in the glycosidic bond are shifted strongly downfield (up to 9ppm) compared to the corresponding unlinked carbons.

This invention provides, in some embodiments, a composition comprisingβ-1-6-glucan, wherein the glucan is conjugated to a solid support. Inone embodiment, the solid support is a bead or particle.

In one embodiment, the beads or particles to which glucans areconjugated comprise denatured proteins (e.g. human serum albumin(Benacerraf et al., 1957 Brit. J. Exp. Path, 38:35)), insolublematerials (e.g. carbon black, silica, silicon dioxide, polystyrene,latex), metal oxides (e.g. titanium oxides, iron oxides), and India ink(i.e., suspension of colloidal carbon particles) (described in Reichardand Filkins, 1984, The Reticuloendothelial System; A ComprehensiveTreatise, pp. 73-101 (Plenum Press), and references therein), hydrogels,(for example as described in US Patent Publication No. 20050191361),sepharose or agarose beads or microparticles. In some embodiments thebeads or microparticles are formed from materials that are biodegradableand non-toxic (e.g. a poly(α-hydroxy acid) such aspoly(lactide-co-glycolide), a polyhydroxybutyric acid, a polyorthoester,a polyanhydride, a polycaprolactone, etc.). The beads or particles ofthe present invention may comprise red blood cells (RBCs) that have beenpurged of their cytoplasm, known as ‘Ghost’ RBCs, bacteria (as bacteriaare cleared by the RES; see, e.g. Benacerraf and Miescher, 1960, Ann NYAcad Sci, 88:184-195), cell fragments, liposomes, bacteriophages,bacteriophage fragments, and viral capsids devoid of the viral nucleicacids (e.g. hepatitis B virus surface antigen particles), etc.

In one embodiment, conjugation to the particle or solid support is viachemically cross-linking the particle/solid to the glucans of thisinvention. The chemistry of cross-linking is well known in the art. Thenature of the crosslinking reagent used to conjugate the glucan and thesolid (e.g. bead or particle) can be any suitable reagent known in theart. It is to be understood that any suitable crosslinking agent may beused with care taken that the activity of the glucan is preserved.

In some embodiments, the glucan is linked to a targeting moiety asfurther herein described. In some embodiments, the term “conjugate” or“linked” and grammatical forms thereof refer to any association betweenthe indicated molecules. In some embodiments, such linkage is covalent,and in some embodiments, such linkage is non-covalent. In someembodiments, such linkage is direct, and in some embodiments, is via alinker molecule.

In some embodiments such linkage will be via any means known in the art,and as described herein. For example, and in some embodiments, linkagemay be via amide formation, urethane, imine or disulfide linkage betweenthe respective molecules, or between a linker moiety with the respectivemolecules. It is to be understood that there is no limitation withrespect to the chemical backbone of the linker molecules. In someembodiments, the linker backbone may be biocompatible, non-immunogenicand/or water soluble. In some embodiments, the linker may comprise polyethylene glycol (PEG), further comprising active chemical groups whichfacilitate linkage as herein described.

In some embodiments, other linkers, which may readily be used for suchpurpose comprise alkanes, polyesters, polyimines, poly-acids, proteins,peptides, DNA, RNA, other glucans, lipids, saccharides, polysaccharides,carbon nanotubes, dendrimers, or solid particles, such as, for example,polymers, metals, salts, inorganic materials, etc.

The particle may be a fragment of a bacteriophage or bacteria.

In certain embodiments the size of the particle is appropriate foringestion by macrophages, neutrophils, or both. The particle can haveany of the compositions described herein. In certain embodiments theinvention provides a population of particles, wherein at least 50% ofthe particles have a size appropriate for ingestion by macrophages,neutrophils, or both. The invention provides populations of particles,wherein at least 50%, 75%, or 90% of the particles fall within a desiredsize range. In certain embodiments the desired size ranges within ±10%,±20%, ±30%, ±40%, or ±50% of a given value. The value may be, e.g. 20nm, 100 nm, 500 nm, 1, 5, 10, 20, 50 microns, etc. The particles in anyof these embodiments can have any of the compositions described herein.The population can comprise particles having different compositions, inany ratio. The populations of particles may be used for any of thepurposes described herein, and methods for such use are an aspect ofthis invention.

Cross-linking reagents that can be used include, but are not limited to,p-Azidobenzoyl hydrazide,N-(4-[p-Azidosalicyclamido]-butyl)-3′(2′-pyridyldithio)-propionamide,Bis(beta-[4-azidosalicylamido]-ethyl)disulfide,1,4-bismaleimidyl-2,3-dihydroxybutane, 1,6-Bismaleimidohexane,1,5-Difluoro-2,4-dinitrobenzene, Dimethyl adipimidate-2HCl, Dimethylsuberimidate-2HCl, Dimethyladipodimidate-2HCl, Dimethylpimelimidate-2HCl, Disuccinimidyl glutarate, Disuccinimidyl tartrate,1-Ethyl-3-[3-Dimethylanonopropyl]Carbodiimide Hydrochloride, (N-Hydroxysuccinimidyl)-4-Azidosalicylic acid, Sulfosuccinimdyl2-[7-azido-4-methyl-coumarin-3-acetamidomethyl-1,3-aminopropionate,N-Succinimidyl-4-iodoacetylaminobenzoate,N-Succinimidyl-3-[2-pyridylthio]propionate, and Succinimidyl6-[3-(2-pyridylathio)-propionamide]hexanoate (Pierce Chemical Co.,Rockford, Ill.) In one embodiment, the glucans are derivatized asdescribed in Nature Methods Vol 2 No. 11, p., 845, 2005, or a similarapproach. In one embodiment glucans are derivatized with a moiety thatprovides a free, reactive primary amine using a reagent such as2,6-diaminopyridine (DAP). The Schiff base azomethine can be reduced,e.g. by sodium cyanoborohydride to a stable secondary amine. In oneembodiment, the derivatized glucan is then reacted with anN-hydroxysuccinimide (NHS) ester, such as NHS-biotin.

Other crosslinking reagents comprise aldehyde, imide, cyano, halogen,carboxyl, activated carboxyl, anhydride and maleimide functional groups.In some embodiments, the cross-linking reagent may compriseheterobifunctional crosslinking reagents such as ABH, M2C2H, MPBH andPDPH (Pierce Chemical Co., Rockford, Ill.). See, e.g. Hermanson, G. T.(1996). Bioconjugate Techniques, Academic Press, Inc., for furtherdiscussion of cross-linking methods and reagents.

In another embodiment, conjugation of the glucan to the beads orparticles may be via use of beads comprising functional groups which canbe conjugated according to methods as disclosed by, e.g. Brumeanu et al.(Genetic Engineering News, Oct. 1, 1995, p. 16).

It is also possible to conjugate the beads/particles/solid/targetingmoiety to the glucan by non-covalent means. One convenient way forachieving non-covalent conjugation comprises utilizing antibodies to theglucan, which are covalently or non-covalently attached to the particle,bead, etc. In another embodiment, non-covalent conjugation is achievedusing biotin-avidin (where “avidin” should be understood to refer to anyform of avidin). For example, avidin-coated or conjugated beads may becontacted with glucan derivatized with a biotin moiety.

In some embodiments, preparation of the conjugated glucans includespurification of the final conjugate substantially free of unconjugatedreactants. Purification may be achieved by affinity, gel filtration,hydrophobic chromatography, tangential ultrafiltration, diafiltration orion exchange chromatography based on the properties of either componentof the conjugate. For example, purification may reduce the amount of oneor more of the unconjugated reactants (e.g. glucan or solid support) to10% or less, 5% or less, or 1% or less of the amount of unconjugatedreactant that was originally present.

In some embodiments, the invention provides a particle comprisingβ-1-6-glucan, which in some embodiments, is enriched for O-acetylatedgroups. In some embodiments, the particle comprises at least 50%β-1-6-glucan by weight. In some embodiments, the β-1-6-glucan ishomogeneously distributed in the particle. It is to be understood thatthe particles comprising β-1-6-glucan of this invention, may in turnencompass any embodiment appropriate thereto, as described herein.

In one embodiment, the conjugated glucan is enriched for O-acetylatedgroups, and in one embodiment, contains at least 25% by weightO-acetylated glucan, or any related embodiment as herein described. Inone embodiment, the glucan is conjugated to a microsphere, which, in oneembodiment, has a diameter of about 1-100 microns. In one embodiment,the microsphere has a diameter which ranges from about 10-50 microns. Inanother embodiment, the microsphere has a diameter which ranges fromabout 5-40 microns. In another embodiment the diameter ranges from 0.1to 5 microns. In another embodiment the diameter ranges from 0.5 to 1micron. In another embodiment, the particle or bead is in the nanometerrange, e.g. 100 to 500 nm.

In one embodiment, the term “bead” or “particle” or “solid support”refers to a material, which is spherical. In another embodiment, term“bead” or “particle” or “solid support” refers to a material, which isnon-spherical. In one embodiment, non-spherical beads or particlespossess a longest axis or longest dimension between any two points ontheir surface within any of the afore-mentioned ranges. In oneembodiment, the dimensions of the particle (e.g. diameter) are selectedto promote phagocytosis of the particles by phagocytic cells, such asneutrophils, macrophages or dendritic cells.

In one embodiment, the term “bead” or “particle” or “solid support”refers to any solid or gelled, or sol-gel-based material, to which theglucan can be adhered, of a size and composition, which can be taken upby phagocytic cells.

In one embodiment, the compositions of this invention comprise ormethods of this invention make use of beads or particles havingdimensions and surface density of glucan (e.g. β-1,6-glucan, optionallyenriched for O-acetylated groups), that is efficiently phagocytosed byantigen-presenting cells as compared, e.g. with particles havingdifferent dimensions and/or surface density of glucan.

In one embodiment, conjugation to the solid support may be accomplishedwith a direct linkage via reaction with solid supports comprising areactive functional group.

Linking chemistries to bind the linker to the β-1-6-glucan and/or tobind the linker to the antibody include, inter alia, amide formation,urethane, imine or disulfide linkage.

The chemical backbone for the linker molecules is not limited. In oneembodiment, the backbone is biocompatible, non-immunogenic andwater-soluble. In one embodiment, the linker is polyethylene glycol(PEG). Other linkers include, inter alia, alkanes, polyesters,polyimines, poly-acids, proteins, peptides, DNA, RNA, glucans, lipids,saccharides, polysaccharides, carbon nanotubes or dendrimers. In oneembodiment, the linker is a solid particle, which may be, inter alia, apolymer, a metal, a salt, a natural material or an inorganic materialsuch as silica.

Linkages via a linker group may be made using any known procedure, forexample, the procedures described, for example, in U.S. Pat. Nos.6,642,363; 4,882,317; or 4,695,624. A useful type of linkage is anadipic acid linker, which may be formed by coupling a free —NH₂ group onan aminated glucan with adipic acid (using, for example, diimideactivation), and then coupling a protein to the resultingsaccharide-adipic acid intermediate. Another type of linkage is acarbonyl linker, which may be formed by reaction of a free hydroxylgroup of a modified glucan with CDI followed by reaction with a proteinto form a carbamate linkage. Other linkers include B-propionamido,nitrophenyl-ethylamine, haloacyl halides, glycosidic linkages,6-aminocaproic acid, ADH, C4 to C12 moieties, etc.

In another embodiment, the invention provides a particle comprising β1-6-glucan. In certain embodiments, the particle consists of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%β-1-6-glucan by dry weight. In certain embodiments, the particleconsists essentially of β-1-6-glucan. In certain embodiments, theparticle consists essentially of β-1-6-glucan, exclusive of any solventcomponent, such as water. In certain embodiments the β-1-6-glucan isenriched for O-acetylated groups. In certain embodiments the particlecontains less than 50%, 40%, 30%, 20%, 10%, or 5% β-1-3 glucan by dryweight. The invention further provides a composition containing any ofthe afore-mentioned particles comprising or consisting essentially ofβ-1-6-glucan, optionally enriched for O-acetylated groups. Thecomposition may further contain a pharmaceutically acceptable carrier oradjuvant. The invention further provides a method of modulating theimmune response of a mammalian subject comprising administering any ofthe afore-mentioned particles, or a composition containing any of theafore-mentioned particles, to the subject. The particle can be preparedusing any method known in the art. The particles can be milled or sievedto achieve a desired size. In certain embodiments the β-1-6-glucan isdistributed evenly, or homogeneously, in the particle. In certainembodiments “distributed evenly” means that the β-1-6-glucan is notencapsulated within a different material, does not simply coat thesurface of a particle comprised of a different material, or is notcovalently or non-covalently attached to the surface of a particlecomposed of a different material. Instead, in certain embodiments theβ-1-6-glucan, optionally mixed with another material, is formed into aparticle such that the β-1-6-glucan is located throughout substantiallythe entire volume of the particle. It will be appreciated that thedensity of the β-1-6-glucan may vary but will generally vary graduallyand continuously throughout the particle rather than abruptly.

In another embodiment, this invention provides a composition comprisinga β-1-6-glucan physically associated with a targeting moiety, which inone embodiment specifically interacts with or attracts a phagocyticcell.

According to this aspect and in one embodiment, the term “physicallyassociated” refers to the formation of a covalent linkage. In oneembodiment, the term “physically associated” refers to strongnon-covalent linkages. In some embodiments, such linkages may beeffected by any means known to one skilled in the art, including someexemplified and described hereinbelow. Linking agents may be readilyapplied for such a purpose, and are commercially available, in someembodiments.

The β-1-6-glucan is linked to a targeting moiety, according to thisaspect of the invention, and in an embodiment thereof. Such a targetingmoiety may comprise any molecule, which specifically interacts with adesired target, which in one embodiment promotes interaction with aphagocytic cell, or, in some embodiments, attracts or recruits aphagocytic cell.

In some embodiments, the targeting moiety is for a particular phagocyticcell type, or in some embodiments, for a particular phagocytic cell, forexample, an infected cell, or in some embodiments, a neoplastic cell orin some embodiments, a preneoplastic cell. In some embodiments, forexample, targeting of a virally infected cell may be accomplished vialinkage of the glucan with a viral co-receptor. In some embodiments,targeting moieties may include integrins or class II molecules of theMHC, which may be up-regulated on infected cells such as professionalantigen-presenting cells.

In some embodiments, targeting of an infected cell results in enhancedtherapeutic responses to infection in the subject. For example, and insome embodiments, targeting the infected cell enhances phagocytosisand/or cytotoxic responses to the pathogen, or in some embodiments,enhances complement-mediated lysis of the pathogen. In some embodiments,targeting of the infected cell enhances the immune response to thepathogen.

In some embodiments, the targeting moiety specifically interacts with aneoplastic or pre-neoplastic cell, as described herein, and comprisingany embodiment thereof. In some embodiments, the use of a β-1-6-glucanlinked to a targeting moiety, which targets a neoplastic orpreneoplastic cell, promotes host anticancer responses. In someembodiments, such targeting promotes tumor cell lysis, or, in someembodiments, enhances host antitumor responses.

In some embodiments, and without limitation, use of the glucans,β-1-6-glucan linked to a targeting moiety and/or compositions of thisinvention are suitable, inter alia, for treating tumors that areresistant to complement-mediated lysis.

In some embodiments, and without limitation, use of the glucans,β-1-6-glucan linked to a targeting moiety and/or compositions of thisinvention target the polysaccharide to an antigen expressed specificallyon cancer cells and thereby enhance complement-mediated lysis of thecells.

In some embodiments, targeting to neoplastic or pre-neoplastic cells ortissue, or tumors can be accomplished by targeting a tumor antigen, asherein described. In some embodiments, such cells may expressadrenomedullin receptors (ADMR), a calcitonin receptor-like receptor(CRLR), CD117 or any combination of tumor-associated antigens, as hereindescribed.

In one embodiment, the targeting moiety is a peptide which binds to anunderglycosylated mucin-1 protein. Mucin-1 (MUC-1) is a transmembranemolecule, which is overexpressed on the cell surface and inintracellular compartments of almost all human epithelial celladenocarcinomas, including more than 90% of human breast cancers,ovarian, pancreatic, colorectal, lung, prostate, colon and gastriccarcinomas. Expression in an underglycosylated form, which exposes animmunogenic epitope that is normally masked, has been demonstrated innon-epithelial cancer cell lines (for example, astrocytoma, melanoma,and neuroblastoma), as well as in hematological malignancies such asmultiple myeloma and some B-cell non-Hodgkin lymphomas, constitutingmore than 50% of all cancers in humans.

According to this aspect, and in one embodiment, by targeting cellsexpressing adrenomedullin receptors or mucin-1 expressing cells with thelinked glucans of this invention, lung, pancreas, ovary, breast andother related cancers may be treated. In some embodiments, by targetingcells expressing CRLR and/or CD117, with the linked glucans of thisinvention, vascular tumors, gliomas, and/or other related cancers may betreated.

In some embodiments, reference herein to a targeting moiety is to beunderstood to encompass an antibody, or fragment thereof as describedherein, a naturally occurring peptide ligand for the referencedreceptor, or a modified form thereof, such as, for example, a truncationproduct. In some embodiments, reference herein to a targeting moiety isto be understood to encompass artificial peptides, small molecules, andthe like.

In some embodiments, many monoclonal antibodies (mAb) are used invarious therapies, which comprise for example, Alemtuzumab (Campath),Bevacizumab (Avastin), Cetuximab (Erbitux), Gemtuzumab (Mylotarg),Ibritumomab (Zevalin), Panitumumab (Vectibix), Rituximab (Rituxan),Tositumomab (Bexxar), Trastuzumab (Herceptin), Palivizumab (Synagis).Any of these mAbs may be linked to a glucan of this invention, orcomprise a composition as herein described, and comprise a targetingmoiety or immune stimulating compound for use in any of the methods asdescribed herein. It is to be understood that any monoclonal antibody orother targeting moiety, or immune stimulating compound may be linked tothe glucans of this invention, or comprise compositions of thisinvention, and such materials are to be considered as part of thisinvention, and encompassed for use in any methods of this invention.

In some embodiments, this invention provides for the use of the glucans,β-1-6-glucans linked to a targeting moiety and/or compositions of thisinvention (as described herein, including any embodiment thereof) as ameans to determine neoplastic or preneoplastic cell or tissueresponsiveness to a treatment regimen. In some embodiments, such methodincludes obtaining a tumor sample from the subject or biopsy materialcontaining the neoploastic or preneoplastic cells and assessing thesensitivity or resistance of the cells to in vitro lysis and/ordetermining the level of expression and/or secretion of an endogenouscomplement control protein.

In some embodiments the tumor cell expresses or overexpresses (e.g.relative to a normal cell of the cell type or tissue of origin of thatcell) an endogenous complement control protein such as complementreceptor 1 (CR1 or CD35), decay-accelerating factor (DAF or CD55),membrane cofactor protein (MCP of CD46), complement factor H (fH) (orFHL-1) and/or C4b-binding protein (C4BP).

In some embodiments, this invention provides for the use of the glucans,β-1-6-glucans linked to a targeting moiety and/or compositions of thisinvention (as described herein, including any embodiment thereof) as ameans to target pathological vasculature, such as, for example,atherosclerotic vasculature, or in some embodiments, targetingpathologic neo-vasculature such as tumor-associated neovasculature forpurposes of enhancing elimination of such vasculature.

According to this aspect of the invention and in some embodiments, thetargeting moiety comprises, inter alia, an antibody or antibody fragmentor ligand specifically interacting with a component of such vasculature,for example, an agent specifically interacting with VEGF, tissue factor,a clotting factor, vascular cell adhesion molecules, integrins,selectins, or any other marker expressed on or at the surface ofendothelial cells.

In one embodiment, the targeting moiety is a peptide, an antibody, anantibody fragment, a receptor, Protein A, Protein G, Protein L, biotin,avidin, streptavidin, a metal ion chelate, an enzyme cofactor, a nucleicacid or a ligand.

In some embodiments, such a targeting moiety may comprise an antibody orantibody fragment. In some embodiments, such an antibody or antibodyfragment will specifically interact with a desired target, as hereindescribed, for example, by interacting with a phagocyte, such thatlinkage of said antibody or fragment with the glucan does not inhibitsuch interaction.

In some embodiments, the term “antibody” refers to intact molecules aswell as functional fragments thereof, such as Fab, F(ab′)2, and Fv thatare capable of specifically interacting with a desired target asdescribed herein, for example, binding to phagocytic cells. In someembodiments, the antibody fragments comprise:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, which can be produced by digestion ofwhole antibody with the enzyme papain to yield an intact light chain anda portion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)2, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

In some embodiments, the antibody fragments may be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can, in some embodiments, be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird el al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

In some embodiments, the antibodies or fragments as described herein maycomprise “humanized forms” of antibodies. In some embodiments, the term“humanized forms of antibodies” refers to non-human (e.g. murine)antibodies, which are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′).sub.2 or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g. mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368 812-13 (1994); Fishwild el al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

In one embodiment, the targeting moiety is an antibody or fragmentthereof, specifically recognized by a neutrophil, for example, andantibody specifically recognizing L-selectin, β2-integrins, complementreceptor 1 (CR-1), decay-accelerating factor (DAF), C5a receptor,intercellular adhesion molecule-1 (ICAM-1), ICAM-3 and others as will beappreciated by one skilled in the art.

In some embodiments, phagocytic cells are targeted or engaged via amolecule interacting with Fc receptors, chemokine receptors, CD40, CD80,CD86, MHC class II molecules, CD69, ADAM8, CD14, CD163, CD33, CD63,CD68, CD74, CHIT1, CHST10, CSF1R, DPP4, FABP4, FCGR1A, ICAM2, IL1R2,ITGA1, ITGA2, S100A8, TNFRSF8, and others as will be appreciated by oneskilled in the art.

In another embodiment, the targeting moiety may be any appropriatemoiety, for example, aptamers, naturally occurring or artificialligands, or engineered binding proteins may comprise the targetingmoieties as described herein, and their physical association with aglucan as herein described can be readily accomplished by any number ofmeans known in the art, including, for example, the methods describedhereinbelow, or variations thereof, to suit the particular nature of thetargeting moiety chosen.

In one embodiment, the targeting moiety enhances attachment to the cell,or, in another embodiment, enhances homing to the cell. In oneembodiment, the targeting moiety enhances attachment following supply ofan energy source. In one embodiment, the targeting moiety is chemicallyattached to the glucan via a chemical cross-linking group, or in anotherembodiment, forms a stable association with the glucan, or, in anotherembodiment, forms an association with the glucan, which dissociatesfollowing changes in environmental conditions, such as, for example,salt concentration or pH.

In one embodiment, the targeting moiety may be an antibody, whichspecifically recognizes a molecule of interest, such as a protein ornucleic acid. In another embodiment, the antibody may specificallyrecognize a reporter molecule attached to a molecule of interest. Inanother embodiment, the targeting moiety may be an antibody fragment,Protein A, Protein G, Protein L, biotin, avidin, streptavidin, a metalion chelate, an enzyme cofactor, or a nucleic acid. In anotherembodiment, the targeting moiety may be a receptor, which binds to acognate ligand of interest, or associated with a cell or molecule ofinterest, or in another embodiment, the targeting moiety may be a ligandwhich is used to attach to a cell via interaction with its cognatereceptor.

Linking the targeting moiety to the glucan of this invention may beaccomplished by any means known in the art, for example as describedfurther herein in Example 7, or for example, as described in U.S. Pat.No. 5,965,714, or United States Patent Publication No. 20070141084, orSchneerson et al., Proc Natl Acad Sci USA. 2003 Jul. 22; 100(15):8945-50, Lees el al., Vaccine. 1996 February; 14 (3):190-8, or viathe use of a cross-linking agent as described herein, or other methods,as will be appreciated by one skilled in the art.

In some embodiments, glycosylated antibodies are used and theβ-1,6-glucan is linked to the glycosylated residue of the antibody, orin another embodiment, linkages may be multiple and involve multiplesites on the antibody, or targeting moiety, as will be understood by oneskilled in the art.

In some embodiments, linking the glucan to a targeting moiety results inenhanced phagocytosis and/or killing of the targeted cell or organism.In some embodiments, such lysis may be mediated by any professionalantigen presenting cell or killer cell, such as, for example,neutrophils, macrophages, dendritic cells, natural killer cells,cytotoxic T lymphocytes, and others.

In some embodiments, any O-acetylated glucan may be physicallyassociated with a targeting moiety, and comprise the glucans orcompositions of this invention, representing an embodiment thereof. Useof such O-acetylated glucans, for example β-1,3-glucans which have beenO-acetylated, for modulating immune responses, treating cancer orprecancerous lesions, promoting resolution of infection, or any methodas herein described is to be considered as part of this invention.

In some embodiments, any of the glucan preparations of this inventionmay be linked to a labeling agent, such that detection of the glucan isreadily accomplished. In one embodiment, the term “a labeling agent”refers to a molecule which renders readily detectable that which iscontacted with a labeling agent. In one embodiment, the labeling agentis a marker polypeptide. The marker polypeptide may comprise, forexample, green fluorescent protein (GFP), DS-Red (red fluorescentprotein), secreted alkaline phosphatase (SEAP), beta-galactosidase,luciferase, or any number of other reporter proteins known to oneskilled in the art. In another embodiment, the labeling agent may beconjugated to another molecule which provides greater specificity forthe target to be labeled. For example, and in one embodiment, thelabeling agent is a fluorochrome conjugated to an antibody whichspecifically binds to a given target molecule, or in another embodiment,which specifically binds another antibody bound to a target molecule,such as will be readily appreciated by one skilled in the art. In someembodiments, the glucan linked to an antibody incorporates afluorochrome in the antibody as will be appreciated by one skilled inthe art.

In one embodiment, the glucan is enriched for O-acetylated groups, andin one embodiment, the glucan contains at least 25% by weightO-acetylated glucan. In another embodiment, the glucan is isolated orderived from a lichen or a yeast, which in one embodiment isUmbilicariaceae. In one embodiment, the glucan is chemically synthesizedor acetylated. In another embodiment, the composition further comprisesan adjuvant, an antigen, an immuno-modulatory compound, or a combinationthereof. In another embodiment, the phagocytic cell is a professionalantigen-presenting cell. In another embodiment, the phagocytic cell is aneutrophil.

In one embodiment, the targeting moiety is an antibody or antibodyfragment.

The invention provides a coated material comprising (a) a substrate; and(b) a compound or composition comprising β-1-6-glucan. In certainembodiments the β-1-6-glucan is enriched for O-acetylated glucan. Incertain embodiments, the coated material is in the form of, or is acomponent of, an implant or other surgical or medical device. In certainembodiments the coated material is a coated material described inprovisional patent application U.S. Ser. No. 60/817,075, filed Jun. 29,2006, entitled “Coating of Devices with Effector Compounds”, wherein the“effector compound” is or comprises β-1-6-glucan.

The invention provides implants and surgical or medical devicescomprising a coated material of the invention. As used herein, the term“medical device” encompasses implants and any device used in thesurgical or medical management of a subject, wherein the device iscontacted with or introduced into the body of the subject and typicallyremains in contact with, or at least in part within the body for atleast a period of 2 hours, e.g. at least 4, 8, 12, or 24 hours. Incertain embodiments, the term “device” refers to a complete device orany part or component thereof. For example, in many applications, a partfor a device will be treated in accordance with the present inventionand then later assembled with other parts to form a complete device.

In certain embodiments the period is between 1 day and 1 week, 1-4weeks, 4-8 weeks, 1-6 months, 6-12 months, or longer. In certainembodiments the device is intended to remain in contact with, or withinthe body for the remainder of the subject's life (unless the devicefails or needs to be removed, e.g. as a result of infection). In certainembodiments the invention provides implants and surgical or medicaldevices, such as catheters, indwelling intravenous or arterial lines,stents, and grafts, coated with or otherwise constructed to containand/or release any of the compounds or compositions disclosed hereincomprising β-1-6-glucan. Optionally the β-1-6-glucan is enriched forO-acetylated groups. In certain embodiments the device coated with orotherwise containing β-1-6-glucan is more resistant to biofilm formation(e.g. by a fungus or bacterium) than an otherwise identical device notcoated with or containing the compound or composition. In certainembodiments the implant or other device is as described in provisionalpatent application U.S. Ser. No. 60/817,075, filed Jun. 29, 2006,entitled “Coating of Devices with Effector Compounds” wherein theeffector compound is or comprises β-1-6-glucan.

In certain embodiments the coated material, implant or other device ismanufactured as described in provisional patent application U.S. Ser.No. 60/817,075, filed Jun. 29, 2006, entitled “Coating of Devices withEffector Compounds” wherein the effector compound is or comprisesβ-1-6-glucan.

In one embodiment, the β-1-6-glucan is released slowly, over a course oftime (e.g. over 1 day-1 week, 1-4 weeks, 4-12 weeks, 12-24 weeks, 24-36weeks, 36-48 weeks, etc.). In certain embodiments, by the end of thetime period release has substantially ceased. In certain embodiments, atthe end of the time period the release rate is less than about 5% of thepeak rate of release or less than 5% of the average rate of releaseduring the time period, and/or less than about 5, 10, or 20% of thecompound remains associated with the substrate. In other embodiments,the β-1-6-glucan is rapidly released, e.g. at least about 50% of theβ-1-6-glucan is released during the first 24 hours. In certainembodiments release is minimal over a time period of interest, e.g. 1day-1 week, 1-4 weeks, 4-12 weeks, 12-24 weeks, 24-36 weeks, 36-48weeks, etc.). In certain embodiments at least 50, 60, 70, 80, 90, 95% ofmore of the β-1-6-glucan remains associated with the substrate at theend of a time period of interest. Release can be measured in vitro, e.g.under conditions of salt concentration, pH, and temperature thatapproximate physiological conditions in the body of a mammalian subjectand/or in vivo. In certain embodiments, the rate of release iscontrollable, e.g. by appropriate selection of the components of acoating layer and/or their concentration. In certain embodiments thethickness of a coating layer is selected to achieve release over adesired duration. For example, a device having a coating layer of anappropriate thickness or composition may be selected to provide releasefor the expected duration of use, e.g. the time during which the deviceis expected to be in intermittent or continuous contact with thesubject's body.

Any of the coated materials, implants, or other devices disclosed hereinmay comprise a β-1-6-glucan and any one or more therapeutic agentsuseful in treating a medical condition of the subject. “Medicalcondition” encompasses any acquired or inherited disease, disorder, orinjury, etc., for which medical and/or surgical intervention iswarranted.

Exemplary inventive implants and other surgical or medical devicesinclude cardiovascular devices (e.g. implantable venous catheters,venous ports, tunneled venous catheters, chronic infusion lines orports, including hepatic artery infusion catheters, pacemaker wires,implantable defibrillators); neurologic/neurosurgical devices (e.g.ventricular peritoneal shunts, ventricular atrial shunts, nervestimulator devices, dural patches and implants to prevent epiduralfibrosis post-laminectomy, devices for continuous subarachnoidinfusions); gastrointestinal devices (e.g. chronic indwelling catheters,feeding tubes, portosystemic shunts, shunts for ascites, peritonealimplants for drug delivery, peritoneal dialysis catheters, implantablemeshes for hernias, suspensions or solid implants to prevent surgicaladhesions, including meshes); genitourinary devices (e.g. uterineimplants, including intrauterine devices (IUDs) and devices to preventendometrial hyperplasia, fallopian tubal implants, including reversiblesterilization devices, fallopian tubal stents, artificial sphincters andperiurethral implants for incontinence, ureteric stents, temporary orchronic indwelling catheters, bladder augmentations, or wraps or splintsfor vasovasostomy); phthalmonlogic implants (e.g. Molteno implants andother implants for neovascular glaucoma or other eye disorders, drugeluting contact lenses for pterygiums, splints for faileddacrocystalrhinostomy, drug eluting contact lenses (e.g. for cornealneovascularity), implants for diabetic retinopathy, drug eluting contactlenses for high risk corneal transplants); otolaryngology devices (e.g.ossicular implants, Eustachian tube splints or stents for glue car orchronic otitis); plastic surgery implants, and orthopedic implants (e.g.spinal rods, screws, orthopedic prostheses). Other implantable devicesof interest herein include pumps, e.g. for delivery of insulin, painmedications, etc. The pump may be an intrathecal pump. Also encompassedis any type of prosthesis, e.g. any substitute for a missing body part.Additionally encompassed are materials useful for sutures.

In certain embodiments the implant or other medical or surgical deviceis listed in Hunter, T. B. and Taljanovic, M. S., Glossary of MedicalDevices and Procedures: Abbreviations, Acronyms, and DefinitionsRadiographics. 23:195-213, 2003.), which provides a nonlimiting set ofdefinitions commonly accepted in the art.

In certain embodiments the device comprises a tube-shaped structurehaving a lumen, wherein the wall of the structure has an inner and anouter surface, either or both of which is coated with or otherwiseadapted to comprise and, optionally, release a compound or compositiondisclosed herein comprising β-1,6-glucan.

Implants and other surgical or medical devices may be coated with (orotherwise adapted to comprise and, optionally, release) compositions ofthe present invention in a variety of manners, including for example:(a) by affixing to the implant or device a compound or composition (e.g.by spraying the implant or device with a composition comprising acompound or composition of the invention, by dipping the implant ordevice into a solution comprising the compound or composition of theinvention, or by other covalent or noncovalent means); (b) by coatingthe implant or device with a substance such as a hydrogel which will inturn absorb the inventive compound or composition; (c) by interweaving acompound- or composition-coated thread or other substrate into theimplant or device; (d) by inserting the implant or device into a sleeveor mesh which is comprised of or coated with a compound or compositiondisclosed herein; (e) constructing the implant or device itself with acompound or composition disclosed herein; or (f) by otherwise adaptingthe implant or device to release the compound or composition.

In one embodiment the term “coated” refers to the physical attachment,or, in another embodiment, association of a gel, film, foam, particleand/or composition comprising β-1,6-glucan with at least a portion of asurface of a material whose “coating” is desired. In one embodiment,such coating will comprise less than 1% of an exposed surface of thematerial, or in another embodiment, from 1-10%, or in anotherembodiment, from 1-25%, or in another embodiment, from 1-50%, or inanother embodiment, from 1-75%, or in another embodiment, from 1-100% ofat least one surface of the material.

In one embodiment, application of such “coating” will be in a pattern,or on specific regions of the material to suit a particular purpose. Forexample, and in some embodiments, a tube-shaped device such as acatheter may comprise coating of one material on the lumenally exposedsurface of the tube, for example, a coating comprising ananti-inflammatory or anti-proliferative compound, and, in someembodiments, the tube-shaped device may be coated with a differentmaterial, for example, a coating comprising a □-1,6-glucan which, in oneembodiment, inhibits biofilm formation. In one embodiment, the coatingof a material will be on at least one surface of the material, or inanother embodiment, on two or more surfaces of the material, or inanother embodiment, on every exposed surface of the material, or inanother embodiment, on any surface of the material.

In some embodiments, the term “coated material” applies not only to asurface coating of the material, but is to be understood as encompassingembedding and/or impregnating the material, in whole, or in someembodiments, in part, with the gels, films, foams, particles and/orcompositions described herein comprising β-1,6-glucan. In someembodiments, the embedding and/or impregnating the material may beaccording to a desired pattern and/or design, to suit a particularpurpose or application. In some embodiments, multiple coatings may beimpregnated or embedded in the material, each of which may be appliedaccording to a particular pattern or design, which may be the same, orin another embodiment, different than the patterning of a first coating.

In some embodiments, the embedding and/or impregnating may be to aparticular surface of a material, in a particular pattern and/or design,to suit a particular purpose or application. In some embodiments, theembedding and/or impregnating of the material may be to two or moresurfaces of the material in the particular pattern and/or design, orsuch pattern and/or design may vary as a function of the surface towhich the material is being embedded and/or impregnated within.

The devices are made of any of a variety of different materials, asappropriate for their intended use. In certain embodiments the devicesof this invention can be made at least in part from any suitablethermoplastic or thermosensitive (e.g. thermosetting) polymer. Suitablepolymers include, for instance, silicones and urethanes (e.g.polyurethane). In one embodiment, the substrate, material or device canbe made at least in part from polyvinyl chloride. In certain embodimentsthe device is formed at least in part from polyethylene. One of skill inthe art will appreciate that these materials are commonly used fortubular devices such as catheters.

In certain embodiments, the compound or composition comprising adheressufficiently to the implant or other device during storage and at thetime of introduction to the body so that the device withstands routinehandling (e.g. during insertion) and storage without significant loss ofthe compound or composition, e.g. with loss not exceeding 10%, 20%, or30% of the compound or composition. In certain embodiments the compoundor composition does not significantly degrade during storage, prior toinsertion, or when warmed to body temperature after insertion inside thebody (if this is to be performed). In certain embodiments of theinvention the inventive implant or device provides a uniform,predictable, prolonged release of the inventive compound or compositioninto the fluid or tissue surrounding the implant or device once it hasbeen deployed. In certain embodiments, e.g. for vascular stents or otherdevices that may be exposed to blood, the composition or compound andmaterials used to form a coating do not render the surface thrombogenic(causing blood clots to form), or cause significant turbulence in bloodflow (more than would be expected in the case if the device was uncoatedor its surface did not comprise a compound or composition disclosedherein).

In some embodiments, the term “gel” encompasses its ordinary meaning inthe art. In one embodiment, the term “gel” refers to a compositioncomprising a polymer having a fluidity at room temperature between thatof a liquid and a solid. In some embodiments, the term “gel” refers to asolid or semisolid colloid system formed of a solid continuous phase anda liquid phase (either discontinuous or continuous or mixed), which, insome embodiments, can be identified by its outward gelatinousappearance, and/or exhibits properties of a solid such as plasticity,elasticity, or rigidity. In some embodiments, the liquid phase can be a‘dispersed’ phase, or in other embodiments, continuous. In someembodiments, the gelling component (solid phase) is lipophilic andpresent in concentrations of less than 10, or in another embodiment, 15,or in another embodiment 20, or in another embodiment, 25, or in anotherembodiment, 30, or in another embodiment, 40 percent. In someembodiments, the term “gel” may encompass a silica gel, analuminosilicate gel or other materials, which are primarily solid and/orparticulate, microspheroidal, spheroidal, etc., or described withdescriptive properties, terms, or expressions which indicatesdestruction of the two-phase system, such as, pore volume, porediameter, surface area. In one embodiment, the gel is a hydrogel, which,in certain embodiments comprises at least 70, 80, 90, 95, 98% or morewater by weight. In another embodiment, the gel comprises polymersdispersed in solvents other than water or aqueous solutions.

In some embodiments, the term “foam” encompasses its ordinary meaning inthe art. In some embodiments, the term “foam” refers to a colloidalsuspension of a gas in a liquid. In one embodiment, the term “foam”refers to a composition comprising an internal phase of gas in anexternal phase of a liquid or solid. In a liquid foam, in someembodiments, a colloidal adsorptive agent forms a film that bounds a gasbubble, with the colloidal dimension in the foam affecting the thicknessof the film, not the size of the bubble.

In some embodiments, the term “film” encompasses its ordinary meaning inthe art. In one embodiment, the term “film” refers to a layer ofmaterial whose dimension is restricted in one dimension. In someembodiments, the average thickness of the film is between 10 μm and 100μm. In some embodiments, the average thickness of the film is between 1μm and 10 μm. The thickness of the film can vary or be substantiallyuniform (e.g. varying by less than about 1, 5, or 10% over a surface invarious embodiments).

The invention encompasses precursors to the coated material, e.g.compositions comprising a β-1-6-glucan and a precursor material that canbe used to form a coating layer when applied to a substrate or can beused to impregnate a substrate. Optionally the composition comprises asolvent, e.g. one that evaporates to allow formation of a coating layer.In some embodiments the solvent is an aqueous solvent. In someembodiments the solvent is an organic solvent. In some embodiments, thesolvent is polar, or slightly polar. In some embodiments, the solvent isnon-polar, or essentially non-polar. Suitable solvents may include,inter alia, dimethylsulfoxide (DMSO), acetone, alcohols, methylethylketone, toluene, xylene, N,N-dimethyl formamide (DMF), tetrahydrofuranand the like. In some embodiments, the solvent is water.

Also provided are processes for preparing the coated material, implant,or other device of the invention.

The coated substrates, materials and/or devices of this invention maycomprise metallic, ceramic, or polymeric materials, or a combinationthereof. The substrates, materials and/or devices may have a variety ofphysical properties. For example, they may be flexible such that theyreadily conform or bend to adopt a desired shape or configuration underconditions of use, or they may be rigid such that significant force isrequired to cause an alteration in shape. In some embodiments the devicemaintains its shape when supported at only one point or end. The surfacecould be substantially smooth or could be rough and/or comprisecrevices.

In some embodiments, the metallic materials include metals and alloysbased on titanium (such as nitinol, nickel titanium alloys,thermo-memory alloy materials), stainless steel, tantalum,nickel-chrome, or certain cobalt alloys including cobalt-chromium-nickelalloys such as Elgiloy® and Phynox®. Metallic materials also includeclad composite filaments, such as those disclosed in WO 94/16646.

In some embodiments, the ceramic materials include, but are not limitedto, oxides, carbides, or nitrides of the transition elements such astitanium oxides, hafnium oxides, iridium oxides, chromium oxides,aluminum oxides, and zirconium oxides. Silicon based materials, such assilica, may also be used. Any of these materials may be used to form asubstrate or a part of a device of this invention, and may be coatedwith the gels, foams, films, or other compositions comprising aβ-1,6-glucan, as herein described.

Also provided by the present invention are methods of using the devices.The devices may be used in any manner in which conventional counterparts(e.g. counterparts not comprising and/or coated with a compound orcomposition disclosed herein) are used, such methods being known in theart. Also provided by the present invention are methods of delivering acompound or composition disclosed herein comprising a β-1,6-glucan to asubject, wherein the method comprises implanting or introducing a coatedmaterial or device comprising a compound or composition of the inventioninto the body of the subject.

In another embodiment, any of the compositions of the inventioncomprises an adjuvant, an antigen, an immuno-modulatory compound, or acombination thereof.

In one embodiment, this invention provides for the combined use of, orcompositions comprising β-glucans and an adjuvant. In some embodiments,the adjuvant may include, but is not limited to: (A) aluminium compounds(e.g. aluminium hydroxide, aluminium phosphate, aluminiumhydroxyphosphate, oxyhydroxide, orthophosphate, sulphate, etc. [e.g. seechapters 8 & 9 of ref. 96]), or mixtures of different aluminiumcompounds, with the compounds taking any suitable form (e.g. gel,crystalline, amorphous, etc.), and with adsorption being preferred, (B)MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated intosubmicron particles using a microfluidizer); (C) liposomes; (D) ISCOMs,which may be devoid of additional detergent; (E) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP,either micro fluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion; (F) Ribi™ adjuvant system (RAS), (RibiImmunochem) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (G) saponin adjuvants, suchas QuilA or QS21, also known as Stimulon™; (H) chitosan; (I) completeFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colonystimulating factor, tumor necrosis factor, etc.; (K) monophosphoryllipid A (MPL) or 3-O-deacylated MPL (3dMPL)]; (L) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions; (M)oligonucleotides comprising CpG motifs] i.e. containing at least one CGdinucleotide, with 5-methylcytosine optionally being used in place ofcytosine; (N) a polyoxyethylene ether or a polyoxyethylene ester, (O) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol; (P) an immuno-stimulatory oligonucleotide (e.g. a CpGoligonucleotide) and a saponin; (Q) an immuno-stimulant and a particleof metal salt; (R) a saponin and an oil-in-water emulsion; (S) a saponin(e.g. QS21)+3dMPL+IL12 (optionally+a sterol); (T) E. coli heat-labileenterotoxin (“LT”), or detoxified mutants thereof, such as the K63 orR72 mutants; (U) cholera toxin (“CT”), or diphtheria toxin (“DT”) ordetoxified mutants of either; (V) double-stranded RNA; (W)monophosphoryl lipid A mimics, such as aminoalkyl glucosaminidephosphate derivatives e.g. RC-529]; (X) polyphosphazene (PCPP); or (Y) abioadhesive such as esterified hyaluronic acid microspheres or amucoadhesive such as crosslinked derivatives of poly(acrylic acid),polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose.

Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamineMTP-PE), etc.

In another embodiment, this invention provides for the combined use of,or compositions comprising J-glucans and an antigen.

In various embodiments, the antigen may be any molecule recognized bythe immune system of the subject as foreign. For example, the antigenmay be any foreign molecule, such as a protein (including a modifiedprotein such as a glycoprotein, a mucoprotein, etc.), a nucleic acid, acarbohydrate, a proteoglycan, a lipid, a mucin molecule, or othersimilar molecule, including any combination thereof. The antigen may, inanother embodiment, be a cell or a part thereof, for example, a cellsurface molecule. In another embodiment, the antigen may derive from aninfectious virus, bacteria, fungi, or other organism (e.g. protists), orpart thereof. These infectious organisms may be active, in oneembodiment or inactive, in another embodiment, which may beaccomplished, for example, through exposure to heat or removal of atleast one protein or gene required for replication of the organism. Inone embodiment, the antigenic protein or peptide is isolated, or inanother embodiment, synthesized.

In one embodiment, the term “antigen” refers to a substance such as aprotein, peptide, or any fragment which stimulates or enhances an immuneresponse, following exposure to or contact with the antigen. In oneembodiment, the antigen is a “danger” signal interpreted by the immunesystem of a subject as to initiate or enhance an immune response as aconsequence of the signal. In another embodiment, the antigen representsthe host's ability to distinguish the presence of a molecule which is“non-self”.

In one embodiment, the antigen is derived from a pathogen, an infectedcell, a neoplastic or preneoplastic cell. In another embodiment, theantigen is an autoantigen, or a molecule which initiates or enhances anautoimmune response.

In one embodiment, the antigen is derived from a parasitic agent, whichresides intracellularly during at least some stages of its life cycle.The intracellular parasites contemplated include for example, protozoa.Protozoa, which infect cells, include: parasites of the genus Plasmodium(e.g. Plasmodium falciparum, P. Vivax, P. ovale and P. malariae),Trypanosoma, Toxoplasma, Leishmania, Schistosoma, and Cryptosporidium.In another embodiment the parasitic agent resides extracellularly duringat least part of its life cycle. Examples include nematodes, trematodes(flukes), and cestodes. In some embodiments, the antigen is derived frombyproducts of infection with the protozoa described, for example, eggantigens of the Schistosoma, antigens uniquely expressed from Toxoplasmacysts, and others, as will be appreciated by one skilled in the art.

In one embodiment, the antigen is derived from a diseased and/orabnormal cell. The diseased or abnormal cells contemplated include:infected cells, neoplastic cells, pre-neoplastic cells, inflammatoryfoci, benign tumors or polyps, cafe au lait spots, leukoplakia, otherskin moles, self-reactive cells, including T and/or NK cells, etc

In one embodiment, the antigen is derived from an infectious virusincluding, inter alia, Retroviridae (e.g. human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-HI, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses,human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. Ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses);Arenaviridae (hemorrhagic fever viruses); Reoviridae (erg., reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes viruses); Poxviridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofspongiform encephalopathies, the agent of delta hepatities (thought tobe a defective satellite of hepatitis B virus), the agents of non-A,non-B hepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

In one embodiment, the antigen is derived from bacteria including, interalia, Helicobacter pylori, Borellia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium. M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes.Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Chlamydia sp., Haemophilus influenzae, Bacillus antracis,corynebacterium diphtheriae, corynebacterium sp., Erysipelothrixrhusiopathiae, Clostridium pefringens, Clostridium tetani, Enterobacteraerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroidessp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Actinomyces israelli andFrancisella tularensis.

In one embodiment, the antigen is derived from fungi, including, interalia, Absidia, such as Absidia corymbifera, Ajellomyces, such asAjellomyces capsulatus, Ajellomyces dermatitidis, Arthroderma, such asArthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum,Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii,Aspergillus, such as Aspergillus flavus, Aspergillus fumigatus,Aspergillus niger, Blastomyces, such as Blastomyces dermatitidis,Candida, such as Candida albicans. Candida glabrala, Candidaguilliermondii. Candida krusei, Candida parapsilosis, Candidatropicalis, Candida pelliculosa Cladophialophora, such asCladophialophora carrionii, Coccidioides, such as Coccidioides immitis,Cryptococcus, such as Cryptococcus neoformans, Cunninghamella,Epidermophyton, such as Epidermophyton floccosum, Exophiala, suchExophiala dermatitidis, Filobasidiella, such as Filobasidiellaneoformans, Fonsecaea, such as Fonsecaea pedrosoi, Fusarium, such asFusarium solani, Geotrichum, such as Geotrichum candidum, Histoplasma,such as Histoplasma capsulatum. Hortaea, such as Hortaea werneckii,Issatschenkia, such as Issaischenkia orientalis, Madurella, suchMadurella grisae, Malassezia, such as Malassezia furfir, Malasseziaglobosa, Malassezia obtuse, Malassezia pachvdermatis, Malasseziarestricta, Malassezia sloofiae, Malassezia sympodialis, Microsporum,such as Microsporum canis, Microsporum fulvum, Microsporum gypseum,Mucor, such as Mucor circinelloides, Nectria, such as Nectriahaematococca, Paecilomyces, such as Paecilomyces variotii,Paracoccidioides, such as Paracoccidioides brasiliensis, Penicillium,such as Penicillium marneffei, Pichia, such as Pichia anomala, Pichiaguilliermondii, Pneumocystis, such as Pneumocystis carinii,Pseudallescheria, such as Pseudallescheria boydii, Rhizopus, such asRhizopus oryzae, Rhodotorula, such as Rhodotorula rubra, Scedosporium,such as Scedosporium apiospermum, Schizophyllum, such as Schizophyllumcommune, Sporothrix, such as Sporothrix schenckii, Trichophyton, such asTrichophyton mentagrophytes, Trichophyton rubrum, Trichophylonverrucosum, Trichophylon violaceum, Trichosporon, such as Trichosporonasahii, Trichosporon cutlaneum, Trichosporon inkin, Trichosporonmucoides, or others.

In one embodiment, the pathogenic fungus infects human hosts. In oneembodiment the pathogenic fungus infects non-human animals.

In some embodiments, the compositions and methods of this inventionallow for the combined use of multiple antigens from the same source,multiple antigens from the same class of organism, multiple antigensfrom different classes of organisms, or any combination thereof.

In another embodiment, this invention provides a method of treating,delaying progression of, or reducing the incidence or severity of aninfection in a subject, said method comprising administering to saidsubject a composition comprising purified β-1-6-glucan. In certainembodiments of the invention the infection is one due to a pathogenicfungus. In certain embodiments of the invention the infection is one dueto a pathogenic bacterium, virus, or parasite. In certain embodiments ofthe invention the subject receives, in addition to a composition of thisinvention, any agent known in the art to be useful for treating orpreventing an infection from which the subject is at risk from which thesubject suffers. Thus, in one embodiment, the method comprisesadministering to a subject (i) a composition of this inventioncomprising β-1-6-glucan; and (ii) a known anti-fungal, anti-bacterial,anti-viral, or anti-parasitic agent. The composition and anti-fungalagent could be administered in a single composition or separately. Insome embodiments, such separate administration may be within up to 24 orup to 48 hours apart, and in some embodiments, less than an hour apart.The composition could be suitable for use in humans, for veterinaryapplications, or both.

In some embodiments, the particles, glucans, compositions orcombinations thereof of this invention stimulate, enhance or facilitatecomplement fixation.

According to this aspect, and in some embodiments, the coatedsubstrates, materials, particles, beads, glucans and/or devices of thisinvention may be used in methods of stimulating, enhancing or promotingimmune responses, which involve complement fixation, which result intherapeutic effects in the subject. In some embodiments, such infectionsmay comprise infection with any of the pathogens as herein described. Insome embodiments, such immune response may be directed to sepsis in thesubject. In some embodiments, such immune response may be directed toChagas disease in a subject, a pulmonary pathogen, or a parasite orhelminth. In some embodiments, such immune response is directed againsta viral infection, such as HSV.

In some embodiments, the methods according to this aspect of theinvention may further comprise administration of an agent which promoteselaboration of the complement cascade. In some embodiments, according tothis aspect of the invention, the methods may further compriseadministration of an antibody which specifically recognizes thepathogenic agent with which the subject is infected.

In one embodiment, the β-1-6-glucan is enriched for O-acetylated groups,which in one embodiment contains at least 25% by weight O-acetylatedglucan and certain embodiments contains between 10% and 20%, or between20% and 25% by weight O-acetylated glucan. In another embodiment, thecomposition further comprises an adjuvant, an antigen, a peptide, animmuno-stimulatory compound, a chemotherapeutic or a combinationthereof. In one embodiment, the antigen or peptide is derived from thesource of the infection. In one embodiment, the immuno-stimulatorycompound is a cytokine. In another embodiment, the chemotherapeuticcompound is an antibiotic or antiviral compound.

In another embodiment, this invention provides a method of treating,delaying progression of, prolonging remission of, or reducing theincidence or severity of cancer in a subject, said method comprisingadministering to said subject a composition comprising purifiedβ-1-6-glucan.

In one embodiment, the antigen is a tumor-associated antigen, or inanother embodiment, the peptide is derived from a tumor-associatedantigen.

In one embodiment, the subject has a hyperplastic or preneoplasticlesion. In another embodiment, the subject has cancer.

In one embodiment, cancers associated with the following cancer antigenmay be treated or prevented by the methods and compositions of theinvention. KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J.Immunol. 142:32-37; Bumal, 1988, Hybridoma 7(4):407-415), ovariancarcinoma antigen (CA125) (Yu el at., 1991, Cancer Res. 51(2):48-475),prostatic acid phosphate (Tailor et al., 1990, Nucl. Acids Res.18(1):4928), prostate specific antigen (Henttu and Vihko, 1989, Biochem.Biophys. Res. Comm. 10(2):903-910; Israeli et al., 1993, Cancer Res.53:227-230), melanoma-associated antigen p97 (Estin et al., 1989, J.Natl. Cancer Instit. 81 (6):445-44), melanoma antigen gp75(Vijayasardahl el al., 1990, J. Exp. Med. 171(4):1375-1380), highmolecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer59:55-3; Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144)),prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foonel al., 1994, Proc. Am. Soc. Clin. Oncol. 13:294), polymorphicepithelial mucin antigen, human milk fat globule antigen, colorectaltumor-associated antigens such as: CEA, TAG-72 (Yokata et al., 1992,Cancer Res. 52:3402-3408), C017-1A (Ragnhammar et al., 1993, Int. J.Cancer 53:751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol.2:135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie etal., 1994, Blood 83:1329-1336), human B-lymphoma antigen-CD20 (Reff etal., 1994, Blood 83:435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med.34:422-430), melanoma-specific antigens such as ganglioside GD2 (Salehet al., 1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara etal., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2(Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3(Hoon et al., 1993, Cancer Res. 53:5244-5250), tumor-specifictransplantation type of cell-surface antigen (TSTA) such asvirally-induced tumor antigens including T-antigen DNA tumor viruses andenvelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185HER2), polymorphicepithelial mucin (PEM) (Hilkens el al., 1992, Trends in Bio. Chem. Sci.17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et al.,1989, Science 245:301-304), differentiation antigen (Feizi, 1985, Nature314:53-57) such as I antigen found in fetal erythrocytes and primaryendoderm, 1 (Ma) found in gastric adenocarcinomas, M18 and M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl,VIM-D5, and D156-22 found in colorectal cancer, TRA-1-85 (blood groupH), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E1 series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Lea) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Leb), G49, EGF receptor, (bloodgroup ALeb/Ley) found in colonic adenocarcinoma, 19.9 found in coloncancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found inmelanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, M1:22:25:8 found inembryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cell stageembryos. In another embodiment, the antigen is a T cell receptor derivedpeptide from a cutaneous T cell lymphoma (see Edelson, 1998, The CancerJournal 4:62).

In another embodiment, the antigenic peptide or protein is derived fromHER2/neu or chorio-embryonic antigen (CEA) for suppression/inhibition ofcancers of the breast, ovary, pancreas, colon, prostate, and lung, whichexpress these antigens. Similarly, mucin-type antigens such as MUC-1 canbe used against various carcinomas; the MAGE, BAGE, and Mart-1 antigenscan be used against melanomas. In one embodiment, the methods may betailored to a specific cancer patient, such that the choice of antigenicpeptide or protein is based on which antigen(s) are expressed in thepatient's cancer cells, which may be predetermined by, in otherembodiments, surgical biopsy or blood cell sample followed byimmunohistochemistry.

In another embodiment, this invention provides for the combined use of,or compositions comprising β-glucans and an immuno-modulatory compound.

Examples of useful immuno-modulating proteins include cytokines,chemokines, complement components, immune system accessory and adhesionmolecules and their receptors of human or non-human animal specificity.Useful examples include, but are not limited to: GM-CSF, IL-2, IL-12,OX40, OX40L (gp34), lymphotactin, CD40, and CD40L. Further usefulexamples include, but are not limited to: interleukins for exampleinterleukins 1 to 15, interferons alpha, beta or gamma, tumor necrosisfactor, granulocyte-macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), granulocyte colonystimulating factor (G-CSF), chemokines such as neutrophil activatingprotein (NAP), macrophage chemoattractant and activating factor (MCAF),RANTES, macrophage inflammatory peptides MIP-1a and MIP-1b, complementcomponents and their receptors, or an accessory molecule such as B7.1,B7.2, TRAP, ICAM-1, 2 or 3 and cytokine receptors. OX40 and OX40-ligand(gp34) are further useful examples of immuno-modulatory proteins. It isto be understood that any compound which may enhance, stimulate ormitigate or abrogate an immune response, in concert with the glucans asherein described in a given immune response may be incorporated in thecompositions of this invention, or used in accordance with the methodsof this invention, and is to be considered an embodiment thereof.

In another embodiment, this invention provides for the combined use of,or compositions comprising β-glucans and at least one adjuvant, antigen,immuno-modulatory compound, or a combination thereof. In anotherembodiment, this invention provides for the combined use of, orcompositions comprising β-glucans, which may be derived from multiplesources, combinations of such glucans and two or more adjuvants,antigens immuno-modulatory compounds, or a combination thereof. Theβ-glucans can be any of the β-glucans described herein, in variousembodiments of the invention.

Once formulated, the compositions of the invention can be administereddirectly to the subject. In some embodiments, the subjects to be treatedare animals, including for example, livestock. In some embodiments, theanimals to be treated are humans. In some embodiments, males and/orfemales can be treated with the compositions and/or according to themethods of this invention. In some embodiments, the subjects to betreated are children and/or teenagers, and/or adults.

In one aspect of the present invention, neutrophils induce heat shockprotein (HSP) expression upon exposure to β-glucan. The greater theexposure to β-glucan, the greater the expression of HSP, and downstreamimmune modulation, in some embodiments.

Microspheres coated with β-1,6-glucan as opposed to β-1,3-glucan weremost effective at inducing HSP expression, ROS production, etc.

HSPs are already associated with peptides that could be presented on MHCclass I and II of antigen-presenting cells. In one embodiment, followingrecognition of β-1,6-glucan in a composition and/or according to amethod of this invention, neutrophils in the subject to which the glucanis administered or with which its cells are contacted, express HSPs tosignal to other immune cells, leading to presentation of other antigenson antigen-presenting cells.

In one embodiment, this invention provides a method of modulating animmune response in a subject, the method comprising administering to thesubject a composition comprising β-1-6-glucan enriched for O-acetylatedgroups, or any embodiment thereof as herein described. In anotherembodiment, this invention provides a method of modulating an immuneresponse in a subject, the method comprising administering to thesubject a composition comprising β-1-6-glucan conjugated to a solidsupport, or any embodiment thereof as herein described. In anotherembodiment, this invention provides a method of modulating an immuneresponse in a subject, the method comprising administering to thesubject a composition comprising β-1-6-glucan, or any embodiment thereofas herein described. In one embodiment, β-glucan in the compositioncomprises at least from about 35-99% by weight, or in anotherembodiment, from about 45-99% by weight, or in another embodiment, fromabout 55-99% by weight, or in another embodiment, from about 65-99% byweight, or in another embodiment, from about 75-99% by weight, or inanother embodiment, from about 85-99% by weight, or in anotherembodiment, from about 90-99% by weight, of β-1-6-glucan, as compared toany other β-glucan. In one embodiment, the term “about” refers to avariance of from 1-10%, or in another embodiment, 5-15%, or in anotherembodiment, up to 10%, or in another embodiment, up to 25% variance fromthe indicated values, except where context indicates that the varianceshould not result in a value exceeding 100%.

According to this aspect of the invention, and in one embodiment,modulating the immune response comprises stimulating said immuneresponse, which in one embodiment is an antigen-specific response. Inone embodiment, the composition further comprises an immunostimulatorycompound, or in another embodiment, a chemotherapeutic compound. Inanother embodiment, the immune response is directed against aninfectious agent, a cancer, a preneoplastic lesion or a combinationthereof, and the compositions comprising, or administration ofβ-1,6-glucan is useful in this context. In one embodiment, according tothis aspect of the invention, additional agents may be administered, orin another embodiment, the compositions for use according to thisaspect, may comprise an additional agent, which is useful in thiscontext.

In one embodiment, according to this aspect of the invention, theadditional agent may comprise an anti-inflammatory agent such asbetamethasone, prednisolone, piroxicam, aspirin, flurbiprofen and(+)-N-{4-[3-(4-fluorophenoxy)phenoxy]-2-cyclopenten-1-yl}-N-hyroxyurea;an antiviral such as acyclovir, nelfinavir, or virazole; an antibioticsuch as ampicillin and penicillin G or belonging to the family ofpenicillines, cephalosporins, aminoglycosidics, macrolides, carbapenemand penem, beta-lactam monocyclic, inhibitors of beta-lactamases,tetracyclins, polipeptidic antibiotics, chloramphenicol and derivatives,fusidic acid, lincomicyn, novobiocine, spectinomycin, poly-ethericionophores, quinolones; an anti-infective such as benzalkonium chlorideor chlorhexidine; dapsone, chloramphenicol, neomycin, cefaclor,cefadroxil, cephalexin, cephradine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin including penicillin G and penicillinV, ticarcillin rifampin and tetracycline; an antiinflammatory such asdiflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid,naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,tolmetin, aspirin and salicylates; antifungal such as amphotericin B,glucan synthesis inhibitors such as caspofungin, micafungin, oranidulafungin (LY303366), econazole, terconazolc, fluconazole,voriconazole or griseofulvin; an antiprotozoal such as metronidazole; animidazole-type anti-neoplastic such as tubulazole; an anthelmintic agentsuch as thiabendazole or oxfendazole; an antihistamine such asastemizole, levocabastine, cetirizine, or cinnarizine; a decongestantsuch as pseudoephedrine; antipsychotics such as fluspirilene,penfluridole, risperidone or ziprasidone; an antineoplastic agent suchas platinum compounds (e.g. spiroplatin, cisplatin, and carboplatin),methotrexate, fluorouracil, adriamycin, mitomycin, ansamitocin,bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine,vincristine, busulfan, chlorambucil, melphalan (e.g. PAM, L-PAM orphenylalanine mustard), mercaptopurine, mitotane, procarbazinehydrochloride dactinomycin (actinomycin D), daunorubicin hydrochloride,doxorubicin hydrochloride, paclitaxel and other taxenes, rapamycin,manumycin A, TNP-470, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testolactone, trilostane, amsacrine(m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase, interferon.alpha.-2a, interferon .alpha.-2b, teniposide (VM-26), vinblastinesulfate (VLB), vincristine sulfate, bleomycin sulfate, hydroxyurea,procarbazine, and dacarbazine; a mitotic inhibitor such as etoposide,colchicine, and the vinca alkaloids, a radiopharmaceutical such asradioactive iodine and phosphorus product, or any combination thereof.

In one embodiment, modulating the immune response comprises stimulatingthe immune response, which in one embodiment is an antigen-specificresponse. According to this aspect of the invention and in oneembodiment, the composition further comprises an immuno-stimulatorycompound or in another embodiment, a chemotherapeutic compound. In oneembodiment, the immune response is directed against an infectious agent,a cancer, a preneoplastic lesion or a combination thereof, or anyembodiment, as herein described.

In some embodiments, the compositions and/or methods of this inventionare applied to or useful in stimulating an immune system in anindividual (animal or human) by the oral or parenteral administration ofcompositions containing the 1-glucans as herein described. In someembodiments, such compositions and/or methods are effective in boostingthe immune response, for example, of individuals, or patients, who areinjured, immunocompromised or protein malnourished. An immunocompromisedindividual refers, in some embodiments, to a person who exhibits anattenuated or reduced ability to mount a normal cellular or humoraldefense to challenge by infectious agents, e.g. viruses, bacteria, fungiand protozoa. A protein malnourished individual refers, in someembodiments, to a person who has a serum albumin level of less thanabout 3.2 grams per deciliter (g/dl) and/or unintentional weight loss ofgreater than 10% of usual body weight.

In some embodiments, the compositions and/or methods of this inventionare used to therapeutically or prophylactically treat animals or humanswho are at a heightened risk of infection due to imminent surgery,injury, illness, radiation or chemotherapy, or other condition whichdeleteriously affects the immune system. In some embodiments, thecompositions and/or methods of this invention are used to treat patientswho have a disease or disorder which causes the normal immune responseto be reduced or depressed, such as HIV infection (AIDS) or who arereceiving immunosuppressive therapy (e.g. individuals who are transplantcandidates or have received a transplant, individuals suffering from anautoimmune disease, etc.). In some embodiments, the compositions and/ormethods of this invention are used to pre-initiate a immune response inpatients who are undergoing chemotherapy or radiation therapy, or whoare at a heightened risk for developing secondary infections orpost-operative complications because of a disease, disorder or treatmentresulting in a reduced ability to mobilize the body's normal responsesto infection.

In another embodiment, modulating the immune response comprisesdownmodulating or abrogating the immune response. According to thisaspect, and in one embodiment, the composition further comprises animmunosuppressant. In one embodiment, the immune response is directedagainst an autoantigen or in another embodiment, an allergen, or inanother embodiment, the immune response is directed against transplantedtissue or in another embodiment, transplanted cells.

In one embodiment, an immune response to a particular antigen may beinitially beneficial to the host, such as, for example, a responsedirected against an antigen from a pathogen that has invaded thesubject. In one embodiment, such an immune response may be too robust,however, such that even after the pathogen has been eradicated, orcontrolled, the immune response is sustained and causes damage to thehost, such as, for example, by causing tissue necrosis, in tissue whichformerly was infected with the pathogen. In these and othercircumstances, the compositions and/or methods of this invention may beuseful in downmodulating an immune response, such that the host is notcompromised in any way by the persistence of such an immune response.

In another embodiment, the immune response, whose downmodulation isdesired is host versus graft disease. With the improvement in theefficiency of surgical techniques for transplanting tissues and organssuch as skin, kidney, liver, heart, lung, pancreas and bone marrow tosubjects, perhaps the principal outstanding problem is the immuneresponse mounted by the recipient to the transplanted allograft ororgan, often resulting in rejection. When allogeneic cells or organs aretransplanted into a host (i.e., the donor and recipient are differentindividual from the same species), the host immune system is likely tomount an immune response to foreign antigens in the transplant(host-versus-graft disease) leading to destruction of the transplantedtissue. Accordingly, the compositions and/or methods of this inventionmay be used, in one embodiment, to prevent such rejection oftransplanted tissue or organ.

In another embodiment, the immune response, whose downmodulation isdesired is graft versus host disease (GVHD). GVHD is a potentially fataldisease that occurs when immunologically competent cells are transferredto an allogeneic recipient. In this situation, the donor'simmunocompetent cells may attack tissues in the recipient. Tissues ofthe skin, gut epithelia and liver are frequent targets and may bedestroyed during the course of GVHD. The disease presents an especiallysevere problem when immune tissue is being transplanted, such as in bonemarrow transplantation; but less severe GVHD has also been reported inother cases as well, including heart and liver transplants. Thecompositions and/or methods of this invention may be used, in oneembodiment, to preventing or ameliorating such disease.

In another embodiment, the immune response, whose down-modulation isdesired is any autoimmune response. According to this aspect of theinvention, and in one embodiment, the method comprises administering thedescribed compositions herein to a subject suffering from an autoimmunedisease or disorder.

In one embodiment, the term “autoimmune disease” refers to the presenceof an autoimmune response in a subject. In one embodiment, the term“autoimmune response” refers to an immune response directed against anauto- or self-antigen. In one embodiment, the autoimmune disease isrheumatoid arthritis, multiple sclerosis, diabetes mellitus, myastheniagravis, pernicious anemia, Addison's disease, lupus erythematosus,Reiter's syndrome, atopic dermatitis, psoriasis or Graves disease.

According to this aspect and in some embodiments, the compositions ofthis invention may further comprise an immunosuppressant. In someembodiments, the methods of this invention may make use of concurrent orsubsequent administration of an immunosuppressant.

In one embodiment the autoimmune disease or disorder is associated withexcessive neutrophil activity, neutrophil infiltration, neutrophildegranulation, etc. In one embodiment the disorder is a disorder thataffects the skin, According to this aspect, and in one embodiment, theglucans, compositions, conjugates, particles, micelles, etc., asdescribed herein may be applied directly to the skin.

In one embodiment, the composition further comprises a steroid. In someembodiments, such compositions are useful for down-modulating orabrogating an immune response, and find application in any of theembodiments described herein for downmodulating such responses.

In one embodiment, the term “steroid” refers to naturally occurringsteroids and their derivatives as well as synthetic or semi-syntheticsteroid analogues having steroid-like activity. In one embodiment, thesteroid is a glucocorticoid or corticosteroid. For example, many suchsteroids have a core fused ring structure based oncyclopentanophenanthrene. Examples of specific natural and syntheticsteroids include, but are not limited to: aldosterone, beclomethasone,betamethasone, budesonide, cloprednol, cortisone, cortivazol,deoxycortone, desonide, desoximetasone, dexamethasone,difluorocortolone, fluclorolone, flumethasone, flunisolide,fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone,fluorocortolone, fluorometholone, flurandrenolone, fluticasone,halcinonide, hydrocortisone, icomethasone, meprednisone, 25methylprednisolone, paramethasone, prednisolone, prednisone, tixocortolor triamcinolone, and their respective pharmaceutically acceptable saltsor derivatives. It will be appreciated that combinations of suchsteroids may also be used in accordance with this invention.

In some embodiments, such compositions are useful for stimulating orenhancing an immune response, and find application in any of theembodiments described herein for stimulating or enhancing suchresponses. In one embodiment, the steroid is an androgen, or an androgenreceptor agonist.

In another embodiment the composition comprises β-1,3-glucans havingβ-1,6-glucan branches (also referred to as beta 1,3/1,6,-glucan orbeta-1,6-branched beta-1,3-glucan) wherein at least some of theβ-1,6-glucan branches are enriched for O-acetylated groups. In anotherembodiment the invention provides a composition comprising (i)β-1,6-glucan enriched for O-acetylated groups; and (ii) β-1,6-branchedβ-1,3-glucan. In another embodiment, the composition is substantiallyfree of β-1,3-glucan. In certain embodiments the composition containsless than 75%, or less than 50%, or less than 25%, or less than 10%, orless than 5%, or less than 1%, or less than 0.1% β-1,3-glucan by weight.In certain embodiments less than 50%, or less than 25%, or 10%, or lessthan 5%, or less than 1%, or less than 0.1% of the total glucan in thecomposition, by weight, is β-1,3-glucan.

It is to be understood that the downmodulation of any immune response,via the compositions and/or methods of this invention of this inventionare to be considered as part of this invention, and an embodimentthereof.

In one embodiment, the compositions and/or methods of this inventionstimulate and/or enhance the secretion of substances, which mediate thesuppressive effects. In one embodiment, the compositions and/or methodsof this invention mediate bystander suppression, without a need fordirect cell contact. In one embodiment, the substances mediatingsuppression secreted by the T suppressor cell populations of thisinvention may include IL-10, TGF-β, or a combination thereof.

In another embodiment, modulating the immune response may compriseshifting the cell type participating in the immune response, cellproduct elaborated during the immune response and/or the overallcharacter of the response, for example, shifting a Th1 to Th2 typeresponse, or vice versa. In one embodiment, the methods/compositions ofthis invention provide for eliciting a “Th1” response, in a diseasewhere a so-called “Th2” type response has developed, when thedevelopment of a so-called “Th1” type response is beneficial to thesubject.

In one embodiment, the term “Th2 type response” refers to a pattern ofcytokine expression, elicited by T Helper cells as part of the adaptiveimmune response, which support the development of a robust antibodyresponse. Typically Th2 type responses are beneficial in helminthinfections in a subject, for example. Typically Th2 type responses arerecognized by the production of interleukin-4 or interleukin 10, forexample.

In another embodiment, the term “Th1 type response” refers to a patternof cytokine expression, elicited by T Helper cells as part of theadaptive immune response, which support the development of robustcell-mediated immunity. Typically Th1 type responses are beneficial inintracellular infections in a subject, for example. Typically Th1 typeresponses are recognized by the production of interleukin-2 orinterferon γ, for example.

In another embodiment, the compositions and/or methods of this inventionare useful in modulating the response such that where a Th1 typeresponse has developed, when Th2 type responses provide a morebeneficial outcome to a subject, the methods and/or compositions of thisinvention provide for a shift to the more beneficial cytokine profile.One example would be in leprosy, where the compositions and/or methodsof the present invention stimulate a Th1 cytokine shift, resulting intuberculoid leprosy, as opposed to lepromatous leprosy, a much moresevere form of the disease, associated with Th2 type responses.

In another embodiment, this invention provides a method of inducingexpression of heat shock proteins in a cell, e.g. an antigen-presentingcell, the method comprising contacting the antigen-presenting cell witha composition comprising β-1-6-glucan enriched for O-acetylated groups.

In another embodiment, this invention provides a method of inducingexpression of heat shock proteins in a cell, e.g. an antigen-presentingcell, the method comprising contacting the antigen-presenting cell witha composition comprising β-1-6-glucan conjugated to a solid support.

As exemplified herein, phagocytosis of particles comprisingβ-1-6-glucan, but not compositions comprising β-1-3-glucan, promotedheat shock protein (hsp) induction.

In one embodiment, the cell is a neutrophil. In another embodiment, theantigen-presenting cell is a dendritic cell or a macrophage.

According to this aspect of the invention, and in another embodiment,this invention provides a method of inducing expression of heat shockproteins in cells, e.g. antigen-presenting cells, neutrophils, etc., themethod comprising contacting the antigen-presenting cells/neutrophilswith a composition comprising β-1-6-glucan, wherein at least 25% of theglucose units in at least 5% of the glucan molecules are enriched forO-acetylated groups.

In another embodiment the invention provides a method of stimulating orenhancing antigen presentation, the method comprising contacting anantigen-presenting cell with a composition comprising β-1-6-glucan,wherein said contact promotes or induces antigen presentation by saidantigen-presenting cell.

In one embodiment, phagocytic cells undergo apoptosis following uptakeof the β-1-6-glucan. According to this aspect of the invention, and inone embodiment, the invention provides a method of promoting orstimulating cellular apoptosis, the method comprising contacting a cellwith a composition comprising β-1-6-glucan, wherein the compositioninduces expression of at least one heat shock protein in said cell andsubsequent apoptosis of said cell. According to this aspect and in oneembodiment, the cell is from a subject with an infection or autoimmunedisease, and promoting apoptosis of such cells provides a therapeuticeffect in the subject.

In some embodiments, this invention provides a method of modulatingmacrophage responsiveness comprising contacting macrophages withneutrophils that have been contacted with a composition comprisingβ-1-6-glucan enriched for O-acetylated groups.

In another embodiment, the invention provides a method modulating animmune response in a subject, the method comprising administering to thesubject a composition comprising a β-1-6-glucan physically associatedwith a targeting moiety, wherein the targeting moiety specificallyinteracts with or attracts a phagocytic cell.

In one embodiment, modulating said immune response comprises stimulatingsaid immune response, which in one embodiment is an antigen-specificresponse. In one embodiment, the composition further comprises animmuno-stimulatory compound, or in another embodiment, the compositionfurther comprises a chemotherapeutic compound. In one embodiment, theimmune response is directed against an infectious agent, a cancer, apreneoplastic lesion or a combination thereof, and in anotherembodiment, the immune response is complement-dependent.

In one embodiment, this invention provides a method of treating,delaying progression of, or reducing the incidence or severity of aninfection in a subject, said method comprising administering to saidsubject a composition comprising a β-1-6-glucan physically associatedwith a targeting moiety, wherein the targeting moiety specificallyinteracts with or attracts a phagocytic cell. In one embodiment, thecomposition further comprises an adjuvant, an antigen, a peptide, animmuno-stimulatory compound, a chemotherapeutic or a combinationthereof. In one embodiment, the antigen or peptide is derived from thesource of the infection. In another embodiment, the immuno-stimulatorycompound is a cytokine. In another embodiment, the chemotherapeuticcompound is an antibiotic or antiviral compound.

In one embodiment, this invention provides a method of stimulating orenhancing heat shock protein expression in a cell, the method comprisingcontacting the cell with comprising a β-1-6-glucan physically associatedwith a targeting moiety, wherein the targeting moiety specificallyinteracts with or attracts a phagocytic cell.

In some embodiments, the methods of this invention serve to enhance theactivity of a variety of cells of the immune system cells such asmacrophages, dendritic cells, etc., in some embodiments, in addition to,or in some embodiments, instead of, neutrophils.

In some embodiments, the methods of this invention serve as a generalapproach to promoting cellular cytotoxic effects, via use of a ligand,which serves to target the cell or material against which a cytotoxicresponse is desired, conjugated to a glucan of this invention, which inturn serves, in some embodiments, to promote cytotxicity against thetargeted cell or material.

In some embodiments, the glucans as described herein, compositionscomprising same, and β-1-6-glucan physically associated with a targetingmoiety, and compositions comprising the same may function to enhancecomplement-mediated lysis in a subject. In some embodiments, suchenhancement may involve the phagocytic cell response, for example,enhancing neutrophil or macrophage, or other professionalantigen-presenting cell phagocytosis and cytotoxic responses. In someembodiments, such enhancement may be independent of phagocytic cellinvolvement, for example, by enhancing membrane attack complex formationand/or activity.

In some embodiments, this invention provides a method of treating,delaying progression of, prolonging remission of, or reducing theincidence or severity of cancer in a subject, via contacting a cell in asubject, or administering to the subject a glucan, composition,conjugate, micelle, preparation or particle of this invention.

In some embodiments, the invention is to be understood as encompassingcompositions that comprise, and conjugates comprising any compound thatfacilitates immune cell recruitment, for example cells of the innateimmune response, such as neutrophils. Such a compound is referred toherein as, inter alia, a targeting moiety, which facilitates immune cellactivation, stimulatory immune responses, cytotoxicity against a desiredtarget, which in some embodiments is specifically targeted through thedescribed moiety, for example, via the specificity dictated by antibodyor fragment conjugated or associated with the glucan.

In some embodiments, the invention comprises conjugates and compositionscomprising an antibody with a desired specificity to suit a particularapplication, as described herein, and as will be appreciated by theskilled artisan. In some embodiments, such conjugates andcompositions/preparations as described herein may comprise, inter alia,any complement-fixing compound known in the art. In some embodiments,conjugates and compositions/preparations as described hereinspecifically exclude cobra venom factor, yet make use of any othercomplement-fixing compound. In some embodiments, the conjugates andcompositions/preparations as described herein may comprise anypolysaccharide except dextran. In some embodiments, the conjugates andcompositions/preparations as described herein may comprise any glucan,including, inter alia, acetylated β-1,6-glucan, β-1,6-glucan, mix ofβ-1,3-glucan and β-1,6-glucan, β-1,3-glucan, mix of β-1,3-glucanβ-1,4-glucan, β-1,4-glucan, acetylated glucans, and/or any glucancomprising any other modification. In some embodiments, the conjugatesand compositions/preparations as described herein may comprise anyglucan, including genetically engineered forms, for example, β-glucanssynthesized in bacteria, yeast, mammalian cells, etc., by recombinantmeans.

In some embodiments, the conjugates and compositions/preparations asdescribed herein may be applied for the immunization of a subjectagainst Candida albicans and other fungal infection.

In any of the afore-mentioned embodiments the contacting may occureither outside the body of a subject or within the body. In oneembodiment, cells, such as antigen-presenting cells, which in someembodiments are neutrophils, or macrophages or dendritic cells, areremoved from a subject, contacted with the composition, and thenadministered to the subject at a subsequent point in time. In oneembodiment the cells are contacted with the composition for a timesufficient to induce expression of heat shock proteins. In oneembodiment the cells are contacted with the composition for a timesufficient to induce production of reactive oxygen species. In oneembodiment the subject receives immunosuppressive therapy prior toadministration of the cells. For example, a subject may be in need ofimmunosuppressive therapy for organ transplantation or other purposes,e.g. chemotherapy or radiation therapy for cancer, leukemia, lymphoma,or any type of tumor, wherein the therapy would tend to render theindividual immunocompromised. In one embodiment of the invention, priorto administering the immunosuppressive therapy, immune system cells areremoved from the subject. The cells (which, in some embodiments, areneutrophils or in other embodiments, other immune system cells, such asother professional antigen-presenting cells, such as macrophages ordendritic cells) are contacted outside the body with a composition ofthis invention and are then returned to the subject a suitable period oftime after the subject has received the immunosuppressive therapy. Thesuitable period of time could be, e.g. after the therapy has beenadministered or its cytotoxic effects have diminished, when the subjectis at risk of or exhibits symptoms or signs of infection, etc.

It is to be understood that the methods and/or compositions of thisinvention which by affecting/modulating an immune response, in turnprevent disease, and/or ameliorate disease, and/or alter diseaseprogression are to be considered as part of this invention.

In some embodiments, the term “contacting” or “administering” refers toboth direct and indirect exposure to the indicated material.

In some embodiments, the compositions and/or methods of this inventioncomprise or make use of a non-sterile or sterile carrier or carriers foradministration to cells, tissues or organisms, such as a pharmaceuticalcarrier suitable for administration to an individual. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, and combinations thereof. The formulation should suitthe mode of administration.

The compositions or glucans of this invention may be administered in anyeffective, convenient manner including, for instance, administration byintravascular (i.v.), intramuscular (i.m.), intranasal (i.n.),subcutaneous (s.c.), oral, rectal, intravaginal delivery, or by anymeans in which the glucan/composition can be delivered to tissue (e.g.needle or catheter). Alternatively, topical administration may bedesired for insertion into epithelial cells. Another method ofadministration is via aspiration or aerosol formulation. In someembodiments the glucan is administered by implanting or introducing intothe body of a subject, an implant or other medical or surgical devicethat comprises the glucan, e.g. as a component of a coating layer.

In one embodiment, the invention provides a food supplement comprisingβ-1-6-glucan enriched for O-acetylated groups. In one embodiment, theinvention provides a food product comprising β-1-6-glucan enriched forO-acetylated groups. In another embodiment, the invention provides acosmetic composition comprising β-1-6-glucan enriched for O-acetylatedgroups.

In some embodiments, a food or food product is any substance that issubstantially non-toxic that can be metabolized by an organism to giveenergy and build tissue. In some embodiments, a food or food productdenotes a product intended for ingestion by a mammal, e.g, by humans,which has nutritional value. In some embodiments a food or food productdenotes a product regulated as a food or food product by the U.S. Foodand Drug Administration (FDA). In some embodiments, a food or foodproduct is a product packaged in a container bearing a label indicatingthat the product is a food or food product. In some embodiments, a foodor food product is a product packaged in a container bearing a labelproviding nutritional information regarding the product, such as thecalorie, fat, or protein content, or the content of one or more vitaminsor minerals. In some embodiments a food supplement (also referred to asa “dietary supplement”) is any substance that is added to a food or foodproduct. In some embodiments the food supplement comprises, in additionto a glucan of this invention, one or more essential nutrients, such asvitamins, minerals, and protein. In some embodiments, the foodsupplement is any product intended for ingestion as a supplement to thediet and may comprise, in addition to a glucan of this invention, one ormore vitamins, minerals, herbs, botanicals, and other plant-derivedsubstances; amino acids; and concentrates, metabolites, constituents andextracts of these substances. In some embodiments, the food, foodproduct, food supplement, or cosmetic composition is not intended todiagnose, cure, mitigate, treat, or prevent disease. In someembodiments, the food supplement is provided in a container or otherpackaging material labeled to indicate that the contents are a food ordietary supplement, e.g. in accordance with then current U.S. law and/orFDA guidelines. In some embodiments, the food supplement or productcomprises about 0.01 to 30 w/w % of the glucan, and may additionallycomprise vitamins, oligosaccharides, dietary ingredients, proteins, or acombination thereof.

In some embodiments, the ratio of the components is not fixed, or inother embodiments, such ratio may range from about 0.01 to 30 w/w % per100 w/w %. Examples of food comprising aforementioned glucan of thisinvention therein are various food, beverage, gum, vitamin complex,health improving food and the like.

The composition may additionally comprise one or more than one oforganic acid, such as citric acid, fumaric acid, adipic acid, lacticacid, malic acid; phosphate, such as phosphate, sodium phosphate,potassium phosphate, acid pyrophosphate, polyphosphate; naturalanti-oxidants, such as polyphenol, catechin, alpha-tocopherol, rosemaryextract, vitamin C, licorice root extract, chitosan, tannic acid, phyticacid etc.

In some embodiments, a cosmetic or personal care composition is acomposition that enhances or improves the appearance of at least aportion of the body, e.g. hair, nails, skin, etc. In one embodiment, thecomposition beautifies the body. In one embodiment the compositionrestores a more youthful appearance. In one embodiment, any compositionapplied or delivered to a subject is considered a cosmetic is it isadministered for purposes of enhancing or improving the appearance of atleast a portion of the body, e.g. hair, nails, skin, etc., or forrestoring a more youthful appearance. In some embodiments, the cosmeticor personal care compositions of this invention may comprise anemollient, moisturizing agent, soothing agent, ultraviolet A or Bblocking agent, retinoid, coloring agent, or fragrance, etc. In someembodiments the cosmetic or personal care composition contains, inaddition to a glucan of this invention, any other component recognizedin the art as being useful in providing a beneficial effect to theappearance. In some embodiments, the cosmetic or personal carecomposition is provided in a container labeled to indicate its intendeduse as a cosmetic and/or labeled to indicate that it is for external useonly.

In some embodiments, the compositions of this invention are formulatedas a topical ointment, lotion, gel, or cream containing the activeingredient(s) in an amount of, for example, 0.0001 to 50% w/w, e.g.0.075 to 20% w/w (including active ingredient(s) in a range between 0.1%and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),often 0.2 to 15% w/w and most often 0.5 to 10% w/w). In someembodiments, when formulated in an ointment, the active ingredients maybe employed with either a paraffinic or a water-miscible ointment base.In some embodiments, the active ingredients may be formulated in a creamwith an oil-in-water cream base.

In some embodiments, the aqueous phase of the cream base may include,for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. In some embodiments, thetopical formulations may include a compound that enhances absorption orpenetration of the active ingredient(s) through the skin or otheraffected areas. Examples of such dermal penetration enhancers includedimethyl sulphoxide and related analogs.

In some embodiments, the compositions of this invention may make use ofemulgents and/or emulsion stabilizers, such as, for example, Tween60™,Span80™, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glycerylmono-stearate and/or sodium lauryl sulfate.

In some embodiments, the choice of suitable oils or fats for theformulation is based on achieving the desired cosmetic properties.Creams are generally non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight- or branched-chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used. These may be used alone or incombination depending on the properties required. In some embodiments,high melting point lipids such as white soft paraffin and/or liquidparaffin or other mineral oils are used.

In some embodiments, the compositions are formulated for use as eyedrops wherein the active ingredient(s) is dissolved or suspended in asuitable excipient(s), for example, an aqueous solvent for activeingredient(s) that comprise one or more charges at pH values nearneutrality, e.g. about pH 6-8. In some embodiments, the activeingredient(s) is present in such formulations in a concentration ofabout 0.5-20% w/w, typically about 1-10% w/w, often about 2-5% w/w.

In some embodiments, the compositions of this invention are formulatedfor topical administration in the mouth, and may include lozengescomprising the active ingredient in a flavored basis, which may comprisesucrose and acacia or tragacanth; pastilles comprising the activeingredient(s) in an inert basis such as gelatin and glycerin, or sucroseand acacia, or others; or mouthwashes comprising the active ingredientin a suitable liquid excipient(s), or others as will be appreciated byone skilled in the art.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.01 to 500 microns (includingaverage particle sizes in a range between 0.01 and 500 microns in 0.1micron or other increments, e.g. 0.05, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 20, 25, 30, 35, 50, 75, 100, etc.microns), which is administered by rapid inhalation through the nasalpassage or by inhalation through the mouth so as to reach the alveolarsacs. Suitable micronized formulations include aqueous or oily solutionsor suspensions of the active ingredient(s). Formulations suitable foraerosol, dry powder or tablet administration may be prepared accordingto conventional methods and may be delivered with other therapeuticagents such as compounds heretofore used in the treatment or prophylaxisof viral or other infections as described herein. Such formulation maybe administered, e.g. orally, parenterally (i.v., i.m., s.c.), topicallyor by a buccal route. According to this aspect of the invention, and insome embodiments, the β-1,6-glucan utilized in the composition isenriched for O-acetylated glucan, as herein described, conjugated to aparticle or bead, as herein described, or a combination thereof.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient(s) such excipients asare known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Unit dosage formulations are those containing adaily dose or unit daily sub-dose, as recited herein, or an appropriatefraction thereof, of the active ingredient(s).

In some embodiments, the β-glucans of this invention, in any of itsforms as described herein, will be administered to a subject at a dosageof 0.1 mg to about 50 mg/kg weight of the subject. In some embodiments,the β-glucans of this invention will be administered according to anyregimen, in terms of the number of times per day, duration of time,etc., which may be adjusted as a part of a course of therapy for asubject, as will be appreciated by the skilled artisan.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents or excipients conventional in the art having regard to the typeof formulation in question, for example those suitable for oraladministration may include flavoring agents.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

For administration to mammals, and particularly humans, it is expectedthat in the case of medications, the physician or other qualifiedhealthcare provider may determine the actual dosage and duration oftreatment, which will be most suitable for an individual and can varywith the age, weight and response of the particular individual. It willbe appreciated that in the case of non-prescription (e.g.“over-the-counter”) medications, foods, food products, food supplements,cosmetic and personal care compositions, the amount may be determined atthe discretion of the user, optionally with guidance from the labelingor from an appropriate health care provider or other advisor.

EXAMPLES Materials and Methods Phagocytes

Neutrophils and monocytes were isolated as described [Rubin-Bejerano,I., et al., Proc Natl Acad Sci USA, 2003. 100(19): p. 11007-12] fromfresh human blood collected from healthy volunteers in accordance with aprotocol approved by the MIT Committee on Use of Humans as ExperimentalSubjects.

Preparation of Candida

The Candida strain was the commonly used laboratory strain CAF2-1 (Ca).Candida was grown on standard media (YPD), as described [Sherman,supra]. Overnight cultures were used in all experiments (about 3×10⁸cells/ml), because the Candida albicans population was found to be morehomogenous (contain >99% yeast form cells) than in mid-logarithmicphase.

In order to test how neutrophils recognize fungi, and to avoid anyalteration of fungi by media or neutrophils or manipulation ofneutrophils by the fungi, fungi were inactivated by UV, which kills thecells but does not alter the fungal cell wall structure [Wheeler, R. T.and G. R. Fink, PLoS Pathog, 2006. 2(4): p. e35].

Opsonization of Candida

A pool of fresh human serum was generated from ten healthy volunteers,and was used for all experiments. Fungal cells were pre-opsonized in 50%serum in Dulbecco's phosphate-buffered saline without calcium chlorideand without magnesium chloride (Gibco) for 15 minutes at 37° C. on amixer. Cells were then incubated on ice for 5 minutes, washed twice with0.04 mg/ml of the protease inhibitor AEBSF (Sigma) in the same buffer,and then washed twice with the same buffer without AEBSF. Fungal cellswere recounted.

Co-Incubation of Phagocytes with Candida

Neutrophils or monocytes were mixed with Candida albicans cells at ratioof 1:5 phagocyte:target. Phagocytes were cultured with opsonized fungi,or alone in RPMI1640 at 37° C. for 2 hours, and were frozen in TRIreagent (MRC) at −80° C.

Microarray Procedure

Total RNA was prepared following the TRI reagent protocol, except thatfor RNA precipitation it was incubated with isopropanol overnight at 4°C. First and second strand synthesis, in vitro transcription,hybridization, and scanning were done as described before[Rubin-Bejerano, I., et al., Proc Natl Acad Sci USA, 2003. 100(19): p.11007-12]. The microarrays were GeneChip Human Genome U133A 2.0 array(Affymetrix).

Microarray Analysis

Data sets were normalized and floored to 20. Ratios of expression fromneutrophils cultured with Candida divided by that from the neutrophilsalone control were calculated. Induced and repressed genes were definedas those with an expression ratio greater than two standard deviationsfrom the mean for a given experiment. Only genes that were consistentlyinduced or repressed in two experimental duplicates were considered asinduced or repressed.

Real Time-PCR (RT-PCR) on Neutrophils Engulfing Candida

Total RNA was prepared following the TRI reagent protocol, except thatfor RNA precipitation it was incubated with isopropanol overnight at 4°C. cDNA was created using High Capacity cDNA Archive Kit (AppliedBiosystems). Quantitative RT-PCR was done using TaqMan® Gene ExpressionAssays (Applied Biosystems) and 7500 Real Time PCR system (AppliedBiosystems), following the manufacturer protocol. The following TaqMan®Gene Expression Assays were used: ACTB (Hs99999903_ml), DNAJB1(Hs00428680_ml), HSPCB (Hs00607336_gH), HSPH1 (Hs00198379_ml), CXCL2(Hs00236966_ml), and CCL3 (Hs00234142_ml). Fold induction was calculatedas the ratio of expression from phagocytes cultured in an experimentalcondition divided by that from the phagocytes alone control.

Carbohydrates

Laminarin (Sigma) is a pure β-1,3-glucan preparation, and Pustulan(Calbiochem) is a pure β-1,6-glucan preparation. Dextran (Fluka) is anβ-1,6-glucan. Glucan from barley (Sigma) is composed of β-1,3-glucan(30%), and β-1,4-glucan (70%). Pustulan was processed as described[Lindberg, B. and J. McPherson, Acta Chem. Scand., 1954. 8: p.985-988.]. When indicated, pustulan was specifically digested using anendo jβ-1,6-glucanase [Lora, J. M., De la Cruz, J., Llobell, A.,Benitez, T. and Pintor-Toro, J. A. Mol Gen Genet. 1995. 247: p.639-45.]. The enzyme was a kind gift from Dr. Nick Zecherle (BiomarinPharmaceutical, Inc). The reaction products were analyzed by gelfiltration and thin-layer chromatography.

Gel Filtration Chromatography

Pustulan (20 mg) was applied to a BioGel P6 column (1.5λ120 cm, BioRad,200-400 mesh). The column was equilibrated in 0.1 M acetic acid and runat a constant flow rate of 15 ml/h, 1.5 ml fractions were collected andcarbohydrate was measured by the phenol-sulfuric acid method. Fractionscontaining the peaks were dried twice for complete elimination of aceticacid and suspended in water at 10-20 mg/ml. Cytochrome C was used as amarker of the exclusion volume and glucose as a marker of the inclusionvolume.

Thin-Layer Chromatography (TLC)

Samples (5 μl) were ascended twice on silica gel 60 plates (Merck, 0.25mm), 20 cm length. The solvent system was n-butanol/ethanol/water(5:3:2). The samples and standards were visualized by heating the platesat 80° C. after spraying with phenol-sulfuric acid. Standardgentiooligosaccharides were prepared from partial acid hydrolysate ofpustulan and isolated by BioGel P4 chromatography as described[[Magnelli, P., Cipollo, J. F., and Abeijon, C. (2002). Anal Biochem301, 136-150.].

O-Deacetylation of Pustulan

Pus input Pus coating PMN expression (fold induction) (μg glucose) (μgglucose/ml) DNAJB1 HSPCB HSPH1

indicates data missing or illegible when filed

(3 ml, 15 mg/ml) was adjusted to 0.1 M NaOH and incubated for 1 hr at37° C. and dialyzed against water.

Preparation of Glucan-Coated Microspheres

Polybead polystyrene 6.0 micron microspheres (Polysciences, Inc.) werecoated with polysaccharides as described [Schlesinger, L. S., S. R.Hull, and T. M. Kaufman, J Immunol, 1994. 152(8): p. 4070-9]. Pustulantends to solidify at room temperature.

14,400 37.3 5.2 4.7 8.3 10,800 19.6 4.6 4.9 10.3 7,200 9.0 7.6 7.2 21.03,600 9.7 2.8 2.4 3.9 1,800 6.6 3.3 3.0 4.9 900 6.6 3.3 2.8 5.9

was solubilized in boiling water, and let cool to room temperature priorto its application to beads. Microsphere coating was detected by thephenol-sulfuric acid method, measuring carbohydrates [Duboius, M., etal., Anal. Biochem., 1956. 28: p. 350-356].

Unless stated otherwise, only beads with 6-15 μg glucose per 1 ml (2×10⁸beads) were included in the analysis (see Table 1). Shortpolysaccharides were not found to coat the beads efficiently by thismethod. Moreover, by this method, acetylated glucans coated the beadsbetter than unacetylated glucans. When not acetylated, more of theglucan was added to the beads to achieve similar levels of coating.

Opsonization of Beads

Beads were opsonized as described above for Candida.

Coincubation of Neutrophils with Beads

Beads were coincubated with neutrophils as described above for Candida.

Real Time-PCR of Neutrophils Engulfing Beads

RT-PCR was performed as described above for Candida. Fold induction wascalculated as the ratio of expression from neutrophils cultured withcarbohydrate-coated beads over neutrophils cultured with untreatedbeads.

Reactive Oxygen Species (ROS) Production Assay

ROS production was assayed using DHR123 (Molecular Probes), whichbecomes fluorescent when oxidized²⁸. 5×10⁶ neutrophils were culturedwith indicated beads at ratio of 1:5 in volume of 1 ml for 1 hr at 37°C. 1 μl of DHR123 (D-23806) was added to 200 μl of culture. Followingincubation at room temperature for 30 minutes the cells were assayed byFluorescence-Activated Cell Sorting (FACS).

Identification of Serum Proteins Binding β-1,6-Glucan

Beads were coated with equivalent amounts of β-1,3-glucan andβ-1,6-glucan, and opsonized as described above. Beads were suspended in2% SDS 1M ammonium hydroxide buffer, and incubated at 37° C. for 1 hour.The supernatant was loaded on 4-20% acrylamide SDS gel. The gel wasstained with silver stain, and bands were cut for analysis by mass spec.

Western Analysis of C3

Western analysis of C3. C3 deposition was assayed using monoclonalantibodies directed against the alpha or the beta chain of C3b (RDIReasearch Diagnostics).

Killing Assay

Viability of Candida albicans was determined using XTT as describedbefore (Meshulam, T., Levitz, S. M., Christin, L., and Diamond, R. D.(1995). J Infect Dis 172, 1153-1156).

Preincubation Experiments

Serum was preincubated with equivalent amount of soluble laminarin orpustulan for 5 minutes at 37° C. The serum was then used to opsonizepustulan-coated beads as described above.

CR3 Blocking Ab

Neutrophils were preincubated with anti-human Mac-1 or isotype controlIgG (Bender Med Systems) for 30 minutes on ice before adding toopsonized pustulan-coated beads.

Example 1 β-1,6-Glucan Stimulates Neutrophils

The Candida strain, CAF2-1, was used to analyze the response ofneutrophils to fungi. Microarray experiments showed that neutrophilsphagocytosing Candida express heat shock proteins (HSPs) (The 10 kDaHSPE1, 40 kDa DNAJB1 DNAJB9, 70 kDa HSPA1A and HSPA9B, 90 kDa HSPCA andHSPCB, and 105/110 kDa HSPH1) (Table 2), along with cytokines andchemokines, such as the IL-8 receptor ligand CXCL2, and CCL3.

TABLE 2 Affymetrix Probe No. Set Name Gene Exp. 1 Exp. 2 1 208744_x_atHSPH1 4.3 4.9 1 206976_s_at HSPH1 7.7 9.6 2 205133_s_at HSPE1 7 9.7 3200064_at HSPCB 3 6.7 3 214359_s_at HSPCB 3.1 7.1 4 211969_at HSPCA 2.75.7 4 210211_s_at HSPCA 3 5.2 4 211968_s_at HSPCA 3.6 7.2 5 200690_atHSPA9B 2.8 3.1 6 202581_at HSPA1A 10.8 12 7 202842_s_at DNAJB9 6.8 6.6 7202843_at DNAJB9 9.1 9.7 8 200666_s_at DNAJB1 5.8 10.1 8 200664_s_atDNAJB1 5.9 11.2

Expression of HSPs by neutrophils was corroborated by quantitativeRT-PCR (FIG. 1A).

The induction of HSPs was greater if Candida was first heated to unmaskthe underlying β-glucan (FIG. 1B), suggesting that expression of HSPs isproportional to exposed β-glucan.

To assay which of the glucan components was responsible for thisinduction, neutrophils were presented with various glucan polymers.Glucans in the soluble form elicited only low levels of HSPs inneutrophils (FIG. 1E).

As neutrophils respond best to particulate material, several sources ofβ-glucan were conjugated to 6 micron polystyrene beads, which aresimilar in size to Candida albicans yeast form cells (5μ). β-1,3-glucanand β-1,6-glucan purified from Candida cell walls were utilized (FIG.1C), as well as non-fungal sources of β-1,3-glucan and β-1,6-glucan thathave been used as standards in previous studies (FIG. 1D) [Brown, G. D.et al. Nature 413, 36-7; Palma, A. S. et al. J Biol Chem 281, 5771-9].

Beads coated with pustulan or β-1,6-glucan from Candida albicanselicited high levels of HSPs in neutrophils (FIG. 1C,D), whereas beadscoated with laminarin or β-1,3-glucan from Candida albicans elicitedminimal expression of HSPs (FIG. 1C,D), despite efficient coating of thebeads. β-glucan from barley, which is composed of β-1,3-glucan (30%) andβ-1,4-glucan (70%) also did not elicit HSPs expression (FIG. 1E). Beadscoated with α-1,6-glucan (dextran) did not elicit any response (FIG.1E), suggesting that the neutrophil response is specific to the (iconfiguration. Elicitation of HSPs required heat-labile serumcomponents, as pustulan-coated beads did not elicit HSPs when opsonizedwith heat-inactivated (HI) serum (FIG. 1D). Acetylated β-1,6-glucanelicited higher levels of HSPs than non-acetylated β-1,6-glucan (compareFIG. 1D to FIG. 1C, and to FIG. 2F), with β-1,6-glucan possessinggreater stimulatory capability than β-1,3-glucan (FIG. 1C,D).

Example 2 Elicitation of HSPs by Pustulan is Due to β-1,6-Glucan andO-Acetylated β-1,6-Glucan

To confirm that elicitation by pustulan is due to β-1,6-glucan, thepustulan was digested with an endoglucanase specific for theβ-1,6-glucan linkage [Lora, J. M. et al. Mol Gen Genet 247, 639-45].

The digestion product resulted in ˜80% reduction in elicitation of HSPs(FIG. 2A). Analysis of the digestion products of pustulan by P-6 sizecolumns (see Methods) revealed a major peak of small products (FIG. 2compare B to C), composed of di- and tri-saccharides as determined byTLC (G2-G3, compare lane 2 and 3, FIG. 2D). These short polymers in theG2-G3 fraction did not coat the beads efficiently.

In addition to the expected products of digestion by theβ-1,6-glucanase, which constitute the vast majority of the pustulan,there was a small peak that was resistant to the enzyme (FIG. 2C,designated as Vo), corresponding to 4-5% of the starting material. Thematerial in this peak was recognized by the anti-glucan antibody andelicited expression of the HSPs in the neutrophils. In order todetermine whether the small peak resistant to enzymatic digestion was amodified form of the polymer, e.g. an acetylated form, the product wasdeacetylated.

Deacetylation of pustulan prior to fractionation virtually abolishedthis minor component (FIG. 2E) and rendered this residuum unable toelicit HSPs (FIG. 2F). These experiments show that the pustulan is apolymer of β-1,6-glucose containing a small amount of O-acetylated orotherwise O-modified β-1,6-glucan. The residual induction of HSPs bypustulan digested with the endo-β-1,6-glucanase may be attributed toO-acetylated pustulan that was resistant to the enzyme and coated thebeads efficiently.

Example 3 β-1,6-Glucan Mediates Efficient Phagocytosis and Production ofReactive Oxygen Species by Neutrophils

Polybead polystyrene microspheres (beads) were coated with β-glucans andthen opsonized. Phagocytosis of beads coated with laminarin (FIG. 3A, aand b) or pustulan (FIG. 3A, c and d) was assessed by time-lapsemicroscopy.

Fluorescence-Activated Cell Sorting (FACS) analysis of neutrophilsingesting beads revealed that beads coated with pustulan were moreefficiently internalized than beads coated with laminarin, althoughlaminarin promoted internalization better than uncoated beads (FIG. 3B,compare panel e to c and a, respectively).

Since killing of pathogens by neutrophils depends on a burst of reactiveoxygen species (ROS) [Babior, B. M. et al. J Clin Invest 52, 741-4], ROSinduction by either of the two glucans was evaluated. ROS could not bedetected in neutrophils alone or in neutrophils presented with beadsthat had not been treated (FIG. 3C, red and green, respectively). Lowlevels were detected with beads coated with laminarin, β-1,3-glucan fromCandida albicans or β-glucan from barley (FIG. 3C, blue, brown, andpurple, respectively). However, beads coated with pustulan stimulatedthe generation of significant amounts of ROS (FIG. 3C, light blue). NoROS was detected by adding soluble pustulan (FIG. 3C, pink). These datashow that β-1,6-glucan evokes a massive production of ROS, whereas theresponse to β-1,3-glucan is much lower by comparison.

Similarly, in FIG. 10, β-1,6-glucan is shown to be required forefficient phagocytosis of Candida albicans, production of ROS, andexpression of HSPs, with samples subjected to β-1,6-glucanase digestionshowing reduced efficacy.

Example 4 Complement Deposition on Beads Coated with β-1,6-Glucan

As serum is required for phagocytosis and the induction of HSPs by beadscoated with β-1,6-glucan (FIG. 1D), it was of interest to determinewhether there was a serum component that was differentially deposited onbeads coated with β-1,6-glucan or β-1,3-glucan. Beads were coated withto laminarin or pustulan and opsonized. Bound proteins were removed frombeads and separated by SDS gel electrophoresis. Although a number ofserum proteins bound both β-1,3-glucan or β-1,6-glucan, there were twoprominent proteins that adhered more avidly to β-1,6-glucan (FIG. 4A).These proteins were extracted from the gel and subjected to analysis bymass spectrometry. The peptides from these bands gave masses thatidentified both proteins as C3, suggesting that proteolytic fragments ofC3 are deposited more avidly on β-1,6-glucan than β-1,3-glucan.

Western analysis revealed that indeed β-1,6-glucan was deposited withmore C3 (FIG. 4B). Antibodies specific for the alpha chain detected highmolecular weight bands, including bands the size of the complete C3 orC3b (105 and 115 kDa, respectively), as well as a lower moleculardoublet the size of C3d (31 and 33 kDa, FIG. 4B a). Beads coated withlaminarin had low levels of these C3 fragments (FIG. 4B a). Antibodiesspecific for the C3 beta chain revealed only the high molecular weightC3/C3b (FIG. 4B b). The 75 kDa chain of C3b or iC3b were not detected(FIG. 4B b).

Example 5 Additional Complement Deposition Studies

To assess further the differences in C3 deposition on β-1,6-glucan ascompared with β-1,3-glucan, serum was preincubated with soluble pustulanor laminarin before using it to opsonize beads coated with pustulan.Preincubation of the serum with soluble pustulan eliminated phagocytosis(FIG. 5A compare c and a) and ROS production (FIG. 5B compare red andblue) by neutrophils, suggesting that serum C3 was titrated out by thesoluble β-1,6-glucan. Serum that was preincubated with an equivalentamount of soluble laminarin still mediated phagocytosis (FIG. 5A compareb and a) and ROS production (FIG. 5B compare red and green), suggestingthat β-1,3-glucan did not efficiently block the interaction of C3 withbeads coated with pustulan.

Western analysis revealed that preincubation with soluble pustulaneliminated most of the C3/C3b and C3d deposition on the pustulan-coatedbeads (FIG. 5C compare 1 to 3). Preincubation with laminarin eliminatedthe low molecular weight C3d doublet, but retained the high molecularweight C3/C3b (FIG. 5C compare 2 to 3), suggesting that the remainingC3/C3b was mediating phagocytosis and induction of ROS (FIGS. 5A and B).Preincubation of serum with soluble pustulan reduced Candida killing(FIG. 5D).

Example 6 Role of CR3 in Phagocytosis

Complement receptor 3 (CR3) is known to mediate phagocytosis ofopsonized yeast and the yeast cell wall preparation zymosan, as well asROS production, thusCR3 mediated phagocytosis of beads coated withβ-1,6-glucan. Anti-human CR3 blocking antibodies reduced the extent ofphagocytosis (FIG. 6A compare b to a) and ROS production (FIG. 6Bcompare red to green), suggesting that CR3 recognized C3b proteolyticfragments (C3d) that are deposited on particulate β-1,6-glucan.

Example 7 β-1,6-Glucan Elicits Chemokines in Monocytes

To assess the role of β-1,6-glucans in eliciting chemokines inmonocytes, polybead polystyrene microspheres (beads) were coated withequivalent amounts of the β-1,3-glucan laminarin (lam), or theβ-1,6-glucan pustulan (pus). Beads were opsonized with pooled humanserum. Monocytes were cultured for 2 hours with 5 mg/ml of soluble lamor pus, or with the beads described above. Induction of chemokines wasdetermined by quantitative real-time PCR. Results were averaged andstandard deviations were calculated (FIG. 7).

Example 8 β-1,6-Glucan Conjugates

In order to determine whether the activity of neutrophils may beenhanced, physical linkage of the polysaccharide to targeting agentssuch as monoclonal antibodies with appropriate specificity was pursued.

Under such a scenario, deposition of complement on the β-1,6-glucanpolysaccharide is expected to recruit neutrophils and enhance engulfmentof the whole complex (FIG. 8). Some antibodies are specifically known tobind complement poorly (for example IgG2 and IgG4). The differentialability of human IgG1 and IgG4 to activate complement is determined bythe COOH-terminal sequence of the CH2 domain, and this technology wouldenhance their efficacy.

The linkage between polysaccharide and antibody can be accomplished viaany number of means, including protocols described for linkingpolysaccharides to proteins [e.g. Bystricky S, et al. Glycoconj J. 2000October; 17 (10):677-80; and Tianhong Chen, et al. Langmuir 2003, 19,9382-9386]. Another means of such linkage comprises modifying thereducing end of the polysaccharide to comprise an amino group asdescribed [Xia B, el al. Nat Methods. 2005 November; 2(11):845-50;Valdivia A, et al. J Biotechnol. 2006 Apr. 10; 122(3):326-33]. The aminogroup could then be coupled to the carboxyl terminus of the antibody.

Toward this end, pustulan (a 20 KDa β-1,6-glucan polysaccharide, whichhas 10-20% 0-acetyl groups) was linked to an anti-Candida albicansmonoclonal antibody following previous methods for linking mannan to BSA[Bystricky S, et al. Glycoconj J. 2000 October; 17 (10):677-80]. Theproduct was dialyzed against water, and complexes larger than 100 kDawere isolated using the appropriate cutoff columns (FIG. 9A). A fractionwas isolated that was larger than 100 kDa and exhibited characteristicbands of the antibody and a smear indicative of the presence of thepolysaccharide, indicating the two were in the same fraction. Since themolecular weight of the polysaccharide is 20 Kda, this suggests that thepolysaccharide and the antibody were linked together.

Sugar quantification was accomplished using a described phenol-sulfuricacid method [Duboius, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A.,and Smith, F. (1956). Anal Biochem 28, 350-356]. The presence ofpolysaccharide in the fraction larger than 100 kDa was therebyconfirmed.

The effect of the modified antibody on the production of ROS byneutrophils cultured with Candida albicans was assessed. Neutrophilscultured with Candida albicans in the presence of the untreated antibodyproduced low levels of ROS (FIG. 9B). Antibody covalently bound to thepolysaccharide, however, elicited high ROS production by neutrophils.Mixing untreated antibody with the polysaccharide without covalentlyattaching them did not produce a comparable effect, indicating that thepolysaccharide must be bound to the antibody to elicit ROS production.

Example 9 In Vivo Protection Medicated by β-1,6-Glucan

Since complement activation was mediated by β-1,6-glucan in vitro, itwas of interest to determine whether such protective responses could beelicited in vivo, in mouse models. Toward this end, it was firstdetermined whether murine serum activates complement (FIG. 11). Beadscoated with pustulan (β-1,6-glucan) and opsonized with murine serumshowed complement activation, as compared to those coated with laminarin(β-1,3-glucan). Mice injected with Candida albicans cells (10⁶) iv,followed by injection of 10⁵ β-1,6-glucan-coated beads or untreatedbeads were evaluated for survival, with treatmed mice demonstratedsignificantly prolonged survival as compared to untreated mice.

PLGA beads encapsulating β-1,6-glucan elicited production of reactiveoxygen species and protected mice from systemic fungal infection (FIG.12). PLGA beads encapsulating β-1,6-glucan elicited higher levels ofreactive oxygen species (Panel B) and markedly enhanced survival (panelC) as compared to PLGA beads encapsulating β-1,3-glucan.

Example 10 β-1,6-Glucan Mediated Immunoglobulin Deposition

Since complement activation was mediated by β-1,6-glucan in vitro, itwas of interest to determine whether immunolglobulin deposition waselicited as well. Toward this end, beads treated with pustulan(β-1,6-glucan) and laminarin (β-1,3-glucan), as well as untreated beadswere opsonized and evaluated for IgM and IgG deposition (FIG. 13).

Markedly enhanced IgG deposition occurred on pustulan treated beads, ascompared to laminarin or untreated beads.

Example 11 An Embodiment of the Production of PLGA MicroparticlesContaining β-1,6-Glucan

The solvent evaporation encapsulation method is commonly used to makebiodegradable polymer microparticles of polymers such as PLGA and PLAbecause these poly acids are highly biocompatible and have favorablebiodegradation kinetics. Solvent evaporation encapsulation involvesseveral steps and common variations:

-   -   1) The polymer is dissolved in a water-immiscible solvent    -   2) The medicament is dissolved, dispersed or emulsified in the        polymeric solution    -   3) The resultant solution, dispersion or emulsion is then        emulsified in a continuous aqueous phase forming discrete        droplets    -   4) The water-immiscible solvent diffuses though the water phase        and evaporates at the water-air interface inducing precipitation        of the polymer and encapsulation of the medicament

The solvent must be immiscible with water and be a suitable solvent forthe medicament, or be immiscible with another solvent which is asuitable solvent for the medicament such that a primary emulsion ofpolymer and drug can be made, or there must be another method ofdispersing the medicament in the polymer solution.

Solubility of β-1,6-Glucan in Candidate Solvents

The solubility of β-1,6-glucan was determined in a number of solventspotentially useful in multiple solvent microemulsion protocols by adding˜10 mg/mL of solid β-1,6-glucan to the solvents at room temperature andobserving degree of dissolution. Water miscible solvents include water,DMSO, methanol and ethanol. Water-immiscible solvents include acetone,methylene chloride and ethyl acetate. β-1,6-glucan demonstratedsolubility >10 mg/mL in only DMSO, but was soluble at lowerconcentrations in water.

Generation of β-1,6-Glucan Encapsulating Microparticles with StandardProtocols

Using a standard protocol for a W/O/W emulsion, β-1,6-glucan wasencapsulated using water as the inner phase, methylene chloride as theoil phase and water as the outer phase. Concentrations of 25 mg/mL inthe inner aqueous phase were achieved by first dissolving β-1,6-glucanin DMSO and then adding to water. The resulting particles (labeled 2%)were tested on neturophils and did not demonstrate activity.

Generation of β-1,6-Glucan Encapsulating Nanoparticles withNano-Precipitation

Nanoprecipitation is a simple method which is useful for making polymernanoparticles which entrap drug. Nanoprecipitation techniques typicallyinclude the following steps:

-   -   1.) Dissolution of the polymer and drug in a solvent    -   2.) Slow addition under vigorous mixing of this polymer/drug        solution to a non-solvent. The non-solvent should be miscible        with the solvent, but the polymer and the drug should not be        soluble in the non-solvent

Since β-1,6 glucan and PLGA are highly soluble in DMSO and non-solublein water, nanoparticles over a range of glucan/PLGA ratios should bepossible. The following conditions were tested:

β-1,6-glucan PLGA Size (nm) 90%  10% 35,650 80%  20% 124 50%  50% 19925%  75% 206 10%  90% 194  0% 100% 201

At concentrations above 80% β-1,6-glucan particles would no longer form.The resulting particles were tested on neturophils and did notdemonstrate activity.

Development of β-1,6-Glucan Nanosuspension-Emulsion Protocol:Modification of Standard W/O/W Protocol Generation of β-1,6-GlucanNanosuspension

β-1,6-glucan was dissolved in dimethyl sulfoxide at room temperature (50mg/mL) and diluted with an equal volume of deionized water. This mixturewas added to a solution of PLGA in dichloromethane under sonication witha probe sonicator.

The DMSO/water/glucan solution was miscible with thePLGA/Dichloromethane solution and formed a single phase. WhenDMSO/water/glucan solution was injected into dichloromethane understirring (not sonication) precipitation of the β-1,6-glucan occurred andthe precipitate formed a stringy gel (during nanoprecipitationprocedure). Under sonication the precipitation appeared to generate acloudy solution which was likely a nano suspension or emulsion which wassuspended in a secondary external emulsion.

The suspension generated above was added to a solution of 1% polyvinylalcohol in water and homonogized to generate polymer microparticles.Evaporation of dichloromethane proceeded during 3 hours of stirring atatmospheric conditions.

The resulting particles were washed 3 times with deionozed water andlyophilized to give a dry powder.

Example 12 β-1,6-Glucan Conjugates can Improve Efficacy of MonoclonalAntibodies

Some monoclonal antibodies (mAb) have good affinity to their target, butdo not elicit a good immune response. Some mAb could become moreefficacious if they had better complement-fixing properties. In someembodiments of this invention the above-described limitations in mAb useare addressed in term of changes in effect of conjugating β-1,6-glucanto mAb, for example on their complement-dependent cytotoxicity (CDC)properties.

The β-1,6-glucan polysaccharide is conjugated directly to an Fc portionof a mAb, for example by oxidation of the polysaccharide and/or antibodywith sodium meta-periodate. Various diamine linkers, including PEGdiamines, as well as biotin-avidin/streptavidin-based conjugation, aretested. Conjugates are monitored to ensure that they retain specificityto the target, retain complement-fixing properties of thepolysaccharide, and are soluble.

Conjugates may comprise, for example, an anti-Candida mAb, or other mAbdirected against cell wall structures such as different mannans andglucans are tested.

β-1,6-glucan is conjugated to a set of syngeneic monoclonal antibodieshaving the same specificity to a target cell (same Fab region), butdifferent conserved regions (Fc), which have different complement-fixingproperties. The polysaccharide is conjugated to IgG2a and IgG2b, and thecapacity of these antibodies to carry out CDC and/or ADCC in vitro istested using dyes that are excluded by live cells (such as propidiumiodide) or by radio isotopes that are released by dead cells (such as51-chromium, ToxiLight, which detects adenylate kinase, etc.).

The polysaccharide-conjugated antibodies are compared to the respectiveunconjugated antibodies (mixed with the polysaccharide without covlanetbinding), or the syngeneic IgG, for increased CDC. Antibodies showingenhanced CDC in association with the conjugated polysaccharide aretested in vivo in a model for these cells. Mice are injected with thetarget cells followed by the unconjugated antibodies, the conjugatedantibodies, or isotype control antibodies and monitored for survival.

Conjugation of the polysaccharides to FDA-approved mAb (includingAlemtuzumab (Campath), Bevacizumab (Avastin), Cetuximab (Erbitux),Gemtuzumab (Mylotarg), Ibritumomab (Zevalin), Panitumumab (Vectibix),Rituximab (Rituxan), Tositumomab (Bexxar), Trastuzumab (Herceptin),Palivizumab (Synagis)) are tested, as well, for increased mAb efficacy.Other mAb which may be tested may comprise such mAB, which have beenshown to lack adequate immune stimulation in clinical trials inprotection studies, whose immune stimulatory capacity may be enhanced bytheir conjugation to the glucans of this invention.

Example 13 β-1,6-Glucan Conjugates for Cancer Treatment

Breast cancer is the second leading cause of cancer death among women inthe western world and the leading cause of death among women between theages of 30 and 70. The highest mortality is restricted to patients whoseregional lymph nodes are involved. Early detection, followed by surgery,provides good prognosis. In patients with occult lymph node metastasis,adjuvant chemotherapy or hormonal therapy for breast cancer have provento be effective, yet may patients will succumb to metastasis.

A number of tumor-associated antigens have been described for breastcarcinomas. The MUC-1 mucin, a high molecular weight glycoprotein, ishighly expressed on breast carconimas. BA-46 is atransmembrane-associated glycoprotein of the human milk fat globulemembrane (HMFG) that is overexpressed in human breast carcinomas.Anti-BA-46 radio-conjugated monoclonal antibodies have successfullytargeted human breast tumors transplanted into mice.

The effect of conjugating β-1,6-glucan to MUC-1 and BA-46-derivedpeptides on their ability to induce CTL which preferentially recognizebreast tumor-derived peptides is tested.

The β-1,6-glucan polysaccharide is conjugated directly to the MUC-1 andBA-46-derived peptides. Conjugates are monitored to ensure that theyretain specificity to the target, retain complement-fixing properties ofthe polysaccharide, and are soluble.

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention as set forth in the appended claims.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the claims.

In the claims articles such as “a,”, “an” and “the” mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext Claims or descriptions that include “or” or “and/or” betweenmembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention provides, in various embodiments, all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of thelisted claims is introduced into another claim dependent on the samebase claim unless otherwise indicated or unless it would be evident toone of ordinary skill in the art that a contradiction or inconsistencywould arise. Where elements are presented as lists, e.g. in Markushgroup format or the like, it is to be understood that each subgroup ofthe elements is also disclosed, and any element(s) can be removed fromthe group. It should it be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. For purposes of simplicity thoseembodiments have not in every case been specifically set forth in haecverba herein. Certain claims are presented in dependent form for thesake of convenience, but Applicant reserves the right to rewrite anydependent claim in independent format to include the elements orlimitations of the independent claim and any other claim(s) on whichsuch claim depends, and such rewritten claim is to be consideredequivalent in all respects to the dependent claim in whatever form it isin (either amended or unamended) prior to being rewritten in independentformat.

What is claimed is:
 1. A composition comprising β-1-6-glucan enrichedfor O-acetylated groups.
 2. The composition of claim 1, wherein saidglucan contains at least 25% by weight O-acetylated glucan.
 3. Thecomposition of claim 1, wherein said glucan is isolated or derived froma lichen, a fungus or a yeast.
 4. The composition of claim 3, whereinsaid glucan is isolated or derived from Umbilicariaceae.
 5. Thecomposition of claim 1, wherein said glucan is genetically engineered,or chemically synthesized or acetylated.
 6. The composition of claim 1,further comprising an adjuvant, an antigen, an immuno-modulatorycompound, or a combination thereof.
 7. The composition of claim 1,wherein said glucan is conjugated to a particle.
 8. A method ofmodulating an immune response in a subject, said method comprisingadministering to said subject a composition comprising β-1-6-glucanoptionally enriched for O-acetylated groups.
 9. The method of claim 8,wherein said glucan contains at least 25% by weight O-acetylated glucan.10. The method of claim 8, wherein said glucan is isolated or derivedfrom a lichen or a fungus, wherein said fungus is optionally a yeast.11. The method of claim 10, wherein said glucan is isolated or derivedfrom Umbilicariaceae.
 12. The method of claim 8, wherein saidcomposition further comprises an adjuvant, an antigen, animmuno-modulatory compound, or a combination thereof.
 13. The method ofclaim 8, wherein said glucan is conjugated to a particle.
 14. The methodof claim 8, wherein modulating said immune response comprisesstimulating said immune response.
 15. The method of claim 8, whereinsaid immune response is an antigen-specific response.
 16. The method ofclaim 8, wherein said composition further comprises a chemotherapeuticcompound.
 17. The method of claim 8, wherein said immune response iscomplement-dependent.
 18. The method of claim 8, wherein said immuneresponse is directed against an infectious agent, a cancer, apreneoplastic lesion or a combination thereof.
 19. The method of claim18, wherein said infectious agent causes sepsis.
 20. The method of claim18, wherein said infectious agent is a parasite, helminth, virus orbacteria.
 21. The method of claim 8, wherein modulating said immuneresponse comprises downmodulating or abrogating said immune response.22. The method of claim 21, wherein said composition further comprisesan immunosuppressant.
 23. The method of claim 21, wherein said immuneresponse is directed against an autoantigen or an allergen.
 24. Themethod of claim 21, wherein said immune response is directed againsttransplanted tissue or cells.
 25. A composition comprising β-1-6-glucan,wherein said glucan is conjugated to a particle.
 26. The composition ofclaim 25, wherein said glucan is enriched for O-acetylated groups. 27.The composition of claim 26, wherein said glucan contains at least 25%by weight O-acetylated glucan.
 28. The composition of claim 25, whereinsaid glucan is isolated or derived from a lichen, a yeast or a fungus.29. The composition of claim 28, wherein said glucan is isolated orderived from Umbilicariaceae.
 30. The composition of claim 25, whereinsaid glucan is genetically engineered or chemically synthesized oracetylated.
 31. The composition of claim 25, further comprising anadjuvant, an antigen, an immuno-modulatory compound, or a combinationthereof.
 32. The composition of claim 25, wherein said particle is amicrosphere or nanoparticle.
 33. The composition of claim 32, whereinsaid microsphere has a diameter of about 0.1-15 microns.
 34. A method ofmodulating an immune response in a subject, said method comprisingadministering to said subject a composition comprising β-1-6-glucan,wherein said glucan is conjugated to a particle.
 35. The method of claim34, wherein said glucan contains at least 25% by weight O-acetylatedglucan.
 36. The method of claim 34, wherein said glucan is isolated orderived from a lichen, a yeast or a fungus.
 37. The method of claim 34,wherein said glucan is isolated or derived from Umbilicariaceae.
 38. Themethod of claim 34, wherein said glucan is genetically engineered orchemically synthesized or acetylated.
 39. The method of claim 34,further comprising an adjuvant, an antigen, an immuno-modulatorycompound, or a combination thereof.
 40. The method of claim 34, whereinsaid glucan is conjugated to a solid support.
 41. The method of claim34, wherein modulating said immune response comprises stimulating saidimmune response.
 42. The method of claim 41, wherein said immuneresponse is an antigen-specific response.
 43. The method of claim 41,wherein said composition further comprises an immuno-stimulatorycompound.
 44. The method of claim 41, wherein said composition furthercomprises a chemotherapeutic compound.
 45. The method of claim 44,wherein said immune response is directed against an infectious agent, acancer, a preneoplastic lesion or a combination thereof.
 46. The methodof claim 34, wherein said immune response is complement-dependent. 47.The method of claim 46, wherein said infectious agent causes sepsis. 48.The method of claim 46, wherein said infectious agent is a parasite,helminth, virus or bacteria.
 49. The method of claim 34, whereinmodulating said immune response comprises downmodulating or abrogatingsaid immune response.
 50. The method of claim 49, wherein saidcomposition further comprises an immunosuppressant.
 51. The method ofclaim 49, wherein said immune response is directed against anautoantigen, an allergen, transplanted tissue or cells.
 52. A method oftreating, delaying progression of, prolonging remission of, or reducingthe incidence or severity of cancer in a subject, said method comprisingadministering to said subject a composition comprising purifiedβ-1-6-glucan.
 53. The method of claim 52, wherein said β-1-6-glucan isenriched for O-acetylated groups.
 54. The method of claim 53, whereinsaid glucan contains at least 25% by weight O-acetylated glucan.
 55. Themethod of claim 52, wherein said glucan is isolated or derived from alichen or yeast or fungus.
 56. The method of claim 52, wherein saidglucan is isolated or derived from Umbilicariaceae.
 57. The method ofclaim 52, wherein said glucan is genetically engineered or chemicallysynthesized or acetylated.
 58. The method of claim 52, furthercomprising an adjuvant, an antigen, a peptide, an immuno-stimulatorycompound, a chemotherapeutic or a combination thereof.
 59. The method ofclaim 58, wherein said antigen is a tumor-associated antigen.
 60. Themethod of claim 58, wherein said peptide is derived from atumor-associated antigen.
 61. The method of claim 58, wherein saidimmuno-stimulatory compound is a cytokine.
 62. The method of claim 58,wherein said glucan is conjugated to a particle.
 63. The method of claim52, wherein said subject has a hyperplastic or preneoplastic lesion. 64.The method of claim 52, wherein said glucan is linked to a targetingmoiety.
 65. The method of claim 64, wherein said targeting moietyspecifically interacts with a tumor-associated antigen.
 66. A method oftreating, delaying progression of, or reducing the incidence or severityof an infection in a subject, said method comprising administering tosaid subject a composition comprising purified β-1-6-glucan.
 67. Themethod of claim 66, wherein said β-1-6-glucan is enriched forO-acetylated groups.
 68. The method of claim 67, wherein said glucancontains at least 25% by weight O-acetylated glucan.
 69. The method ofclaim 68, wherein said glucan is isolated or derived from a lichen or ayeast or a fungus.
 70. The method of claim 68, wherein said glucan isisolated or derived from Umbilicariaceae.
 71. The method of claim 68,wherein said glucan is genetically engineered or chemically synthesizedor acetylated.
 72. The method of claim 68, further comprising anadjuvant, an antigen, a peptide, an immuno-stimulatory compound, achemotherapeutic or a combination thereof.
 73. The method of claim 68,wherein said antigen or peptide is derived from the source of saidinfection.
 74. The method of claim 68, wherein said immuno-stimulatorycompound is a cytokine.
 75. The method of claim 68, wherein saidchemotherapeutic compound is an antibiotic or antiviral compound. 76.The method of claim 68, wherein said glucan is attached to a particle.77. The method of claim 66, wherein said immune response iscomplement-dependent.
 78. The method of claim 66, wherein saidinfectious agent causes sepsis.
 79. The method of claim 66, wherein saidinfectious agent is a parasite, helminth, virus or bacteria.
 80. Amethod of stimulating or enhancing heat shock protein expression in acell, the method comprising contacting said cell with a compositioncomprising purified β-1-6-glucan, wherein said β-1-6-glucan is attachedto a particle.
 81. The method of claim 80, wherein said glucan isenriched for O-acetylated glucan.
 82. The method of claim 80, whereinsaid glucan contains at least 25% by weight O-acetylated glucan.
 83. Themethod of claim 80, wherein said glucan is isolated or derived from alichen or yeast or fungus.
 84. The method of claim 80, wherein saidglucan is isolated or derived from Umbilicariaceae.
 85. The method ofclaim 80, wherein said glucan is genetically engineered or chemicallysynthesized or acetylated.
 86. The method of claim 80, wherein said cellis an antigen-presenting cell.
 87. The method of claim 86, wherein saidcell is a neutrophil.
 88. The method of claim 86, wherein said cell isinfected.
 89. A method of stimulating or enhancing heat shock proteinexpression in a cell, the method comprising contacting said cell with acomposition comprising purified β-1-6-glucan, wherein said β-1-6-glucanis enriched for O-acetylated glucan.
 90. The method of claim 89, whereinsaid glucan contains at least 25% by weight O-acetylated glucan.
 91. Themethod of claim 89, wherein said glucan is isolated or derived from alichen or yeast or fungus.
 92. The method of claim 89, wherein saidglucan is isolated or derived from Umbilicariaceae.
 93. The method ofclaim 89, wherein said glucan is genetically engineered or chemicallysynthesized or acetylated
 94. The method of claim 89, wherein said cellis an antigen-presenting cell.
 95. The method of claim 89, wherein saidcell is a neutrophil.
 96. The method of claim 89, wherein said cell isinfected.
 97. A particle comprising β-1-6-glucan.
 98. The particle ofclaim 97, wherein the β-1-6-glucan is enriched for O-acetylated groups.99. The particle of claim 97, wherein the particle comprises at least50% β-1-6-glucan by weight.
 100. The particle of claim 97, wherein theparticle comprises glucan, and wherein at least 50% of the glucan isβ-1-6-glucan
 101. The particle of claim 97, wherein the β-1-6-glucan ishomogeneously distributed in the particle.
 102. The particle of claim97, wherein the particle has a size appropriate for phagocytosis byneutrophils, monocytes, macrophages, or dendritic cells.
 103. A methodof modulating the immune response of immune system cells comprisingcontacting the cells with the particle of claim 97 in an amountsufficient to modulate the immune response.
 104. The method of claim103, wherein said amount is sufficient to enhance phagocytosis, induceproduction of ROS, or induce expression of heat shock proteins.
 105. Amethod of modulating the immune response comprising administering theparticle of claim 97 to a subject in an amount sufficient to modulatethe immune response.
 106. The method of claim 105, wherein said amountis sufficient to enhance phagocytosis, induce production of ROS, orinduce expression of heat shock proteins.
 107. A composition comprisingpurified β-1-6-glucan, wherein the composition is a pharmaceuticalcomposition, a food or food product, a food supplement, or a cosmeticcomposition.
 108. The composition of claim 107, wherein the compositioncomprises β-1-6-glucan that has been processed to increase its abilityto modulate the immune response relative to unprocessed β-1-6-glucan.109. The composition of claim 107, wherein at least 50% of the glucancontained in the composition is β-1-6-glucan.
 110. A method ofmodulating the immune response comprising administering the particle ofclaim 97 to a subject in an amount sufficient to modulate the immuneresponse.
 111. The method of claim 107, wherein said amount issufficient to enhance phagocytosis, induce production of ROS, or induceexpression of heat shock proteins.
 112. A micelle comprisingβ-1,6-glucan, wherein said β-1,6-glucan is optionally enriched forO-acetylated glucan.
 113. A composition comprising β-1,6-glucan and abiodegradable polymer, wherein said biodegradable polymer degrades toform biologically active salicylate or alpha-hydroxy acid moieties andsaid β-1,6-glucan is optionally enriched for O-acetylated glucan.
 114. Aparticle comprising the composition of claim
 113. 115. A medical devicecomprising the composition of claim
 113. 116. A medical device whereinat least a portion of a surface of the implant or device comprisesβ-1-6-glucan, optionally enriched for O-acetylated glucan.
 117. Themedical device of claim 116, wherein at least a portion of a surface iscoated with a composition comprising a β-1-6-glucan, optionally enrichedfor O-acetylated glucan.
 118. The medical device of claim 116, whereinthe device is selected from the group consisting of: catheter, stent,valve, pacemaker, central line, pessary, tube, shunt, feeding tube,drain, and orthopedic hardware devices.
 119. The medical device of claim116, wherein the composition comprises a coating layer comprising apolymer and β-1-6-glucan, optionally enriched for O-acetylated glucan.120. The medical device of claim 116, wherein the composition comprisesa coating layer comprising a polymer and β-1-6-glucan, optionallyenriched for O-acetylated glucan, wherein the polymer is biodegradable.121. A method of treating a subject comprising implanting or introducingthe medical device of claim 116 into the body of a subject in needthereof.
 122. A coated material comprising: (a) a substrate; and (b) acomposition comprising a β-1-6-glucan physically associated with atleast a portion of a surface of said substrate, wherein said compositionis optionally in the form of a gel or film.
 123. The coated material ofclaim 122, wherein the composition comprises a polymer.
 124. The coatedmaterial of claim 122, wherein the composition comprises a biodegradablepolymer.
 125. The coated material of claim 122, wherein the substrate iscomposed at least in part of metal, ceramic, or polymer.
 126. A methodof treating a subject comprising contacting the body of a subject inneed thereof with the coated material of claim
 122. 127. A compositioncomprising a β-1-6-glucan physically associated with a targeting moiety.128. The composition of claim 127, wherein said glucan is enriched forO-acetylated groups.
 129. The composition of claim 128, wherein saidglucan contains at least 25% by weight O-acetylated glucan.
 130. Thecomposition of claim 127, wherein said glucan is isolated or derivedfrom a lichen or a yeast or a fungus.
 131. The composition of claim 130,wherein said glucan is isolated or derived from Umbilicariaceae. 132.The composition of claim 127, wherein said glucan is geneticallyengineered or chemically synthesized or acetylated.
 133. The compositionof claim 127, further comprising an adjuvant, an antigen, animmuno-modulatory compound, or a combination thereof.
 134. Thecomposition of claim 127, wherein said phagocytic cell is a professionalantigen-presenting cell.
 135. The composition of claim 127, wherein saidphagocytic cell is a neutrophil.
 136. The composition of claim 127,wherein said targeting moiety is an antibody or antibody fragment. 137.A method of modulating an immune response in a subject, said methodcomprising administering to said subject the composition of claim 127.138. The method of claim 137, wherein said glucan is conjugated to aparticle.
 139. The method of claim 137, wherein modulating said immuneresponse comprises stimulating said immune response.
 140. The method ofclaim 139, wherein said immune response is an antigen-specific response.141. The method of claim 137, wherein said composition further comprisesan immuno-stimulatory compound.
 142. The method of claim 137, whereinsaid composition further comprises a chemotherapeutic compound.
 143. Themethod of claim 137, wherein said immune response is directed against aninfectious agent, a cancer, a preneoplastic lesion or a combinationthereof.
 144. The method of claim 137, wherein said immune response iscomplement-dependent.
 145. A method of treating, delaying progressionof, or reducing the incidence or severity of an infection in a subject,said method comprising administering to said subject the composition ofclaim
 127. 146. The method of claim 145, further comprising an adjuvant,an antigen, a peptide, an immuno-stimulatory compound, achemotherapeutic or a combination thereof.
 147. The method of claim 146,wherein said antigen or peptide is derived from the source of saidinfection.
 148. The method of claim 146, wherein said immuno-stimulatorycompound is a cytokine.
 149. The method of claim 146, wherein saidchemotherapeutic compound is an antibiotic or antiviral compound.
 150. Amethod of stimulating or enhancing heat shock protein expression in acell, the method comprising contacting said cell with the composition ofclaim 127.